Exploring the Potential of Lecanicillium aphanocladii AUMC-16936 as a Novel Microbial Cell Factory for the Biosynthesis of CuONPs Against Potato Fusarium Wilt

Exploring the Potential of Lecanicillium aphanocladii AUMC-16936 as a Novel Microbial Cell Factory for the Biosynthesis of CuONPs Against Potato Fusarium Wilt | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Exploring the Potential of Lecanicillium aphanocladii AUMC-16936 as a Novel Microbial Cell Factory for the Biosynthesis of CuONPs Against Potato Fusarium Wilt Noha A. El-Sebaii, Fatmah A. Safhi, Mohamed ا=Halawa, Mai A.El-Esawy, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8360033/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 09 Apr, 2026 Read the published version in Microbial Cell Factories → Version 1 posted 18 You are reading this latest preprint version Abstract Background Utilizing fungal metabolic processes for the eco-friendly synthesis of nanomaterials has arisen as a sustainable substitute for chemical manufacturing. To the best of our knowledge, this is the first report describing the mycosynthesis of bioactive CuONPs using Lecanicillium aphanocladii and evaluating their efficacy against potato Fusarium wilt. Results Herein, CuONPs were mycosynthesized utilizing the cell-free filtrate of L. aphanocladii AUMC-16936, acting as a bio-reductant and capping agent. UV–visible spectroscopy (UV–Vis), Fourier transform infrared spectroscopy (FT-IR), Transmission electron microscopy (TEM), and X-ray spectroscopy (XRD) patterns confirmed the precise composition of the rod-shaped CuONPs with a mean particle size of 15.24 ± 4.19 nm. The biogenic CuONPs demonstrated potent antifungal activity in vitro against Fusarium oxysporum , with 100 ppm identified as the optimal inhibitory concentration. To validate the biological functionality of the synthesized particles, in vivo trials were conducted on potato plants. Foliar application of the mycosynthesized CuONPs (100 ppm) proved superior to soil watering, significantly mitigating Fusarium wilt severity. The treatment triggered a systemic defense response, evidenced by the upregulation of antioxidant enzymes as ascorbate peroxidase (APX), peroxidase (POD), and polyphenol oxidase (PPO), along with non-enzymatic indicators including total antioxidant capacity (TAC), total phenolic content, ascorbic acid, as well as glutathione, leading to a marked reduction in oxidative stress markers (H 2 O 2 , MDA). Moreover, elevated expression levels of several regulatory defense genes, such as PR-1b , IbBBx24 , and CHI , were noted in all plants that have been treated in comparison to infected ones. Conclusions L. aphanocladii AUMC-16936 efficiently yields bioactive CuONPs that combat Fusarium wilt in potato plants offering a promising strategy for sustainable nano-fungicides. Mycosynthesis nanoparticles Lecanicillium aphanocladii Copper oxide Fusarium oxysporum Potato Fausarium wilt Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 Introduction Potatoes ( Solanum tuberosum ) can significantly contribute to food security by improving availability, access, utilization, and stability [ 1 ].Potato production ranks as the third most significant food crop following wheat and rice, sustaining over 1.3 billion individuals [ 2 , 3 ]. However, potato farming encounters considerable obstacles, including fungal infections like Fusarium oxysporum , responsible for Fusarium wilt [ 4 ]. This soil-borne disease can cause significant yield reductions, jeopardizing food security and farmers' livelihoods. F. oxysporum is a notable threat because of its ability to infect potato plants over their whole growth cycle, from germination to maturity [ 5 , 6 ]. It infects plants via root systems, injuries, or natural apertures, resulting in vascular wilt, root rot, and ultimately the decline of potato crops [ 7 ]. The fungus infiltrates the plant's vascular system, resulting in obstructions that hinder the proper water movement and nutrients. Infected plants frequently have dark-brown streaks within the xylem vessels, a characteristic indicative of Fusarium wilt [ 8 , 9 ]. A particularly alarming feature of F. oxysporum infection is the synthesis of mycotoxins, deleterious secondary metabolites that not only intensify plant damage but also threaten the health of humans and animals upon the consumption of infected tubers [ 10 , 11 ]. Existing control strategies predominantly depend on chemical fungicides, crop rotation, and the utilization of disease-resistant potato cultivars [ 12 ]. The excessive application of chemical treatments has resulted in the development of resistant fungus strains and environmental pollution [ 13 ]. The main consequence is that fungicides produce chemical traces on non-target organisms and enter the food chain [ 14 ]. Consequently, these problems highlight the necessity for sustainable, environmentally friendly techniques to effectively address F. oxysporum infections [ 15 ]. By tackling the difficulties presented by fungal infections with innovative and sustainable solutions, the agricultural sector can enhance the resilience and production of potato crops, thereby preserving their essential role in global food chains. Nanotechnology has arisen as a promising solution in the controlling the spread of agricultural diseases [ 16 – 18 ]. Copper Oxide nanoparticles exhibit promising antifungal capabilities simply because of their diminutive size, enormous surface area, and power to produce reactive oxygen species (ROS). These nanoparticles compromise the integrity of the cell wall and membrane of fungal pathogens, impede enzyme functions, and disrupt metabolic pathways, ultimately resulting in the suppression of infections [ 19 , 20 ]. In green chemistry for nanoparticle synthesis, environmentally benign solvents, non-toxic reducing substances, and safe stabilizers are prioritized. This method utilizes plant extracts, fungi, algae, bacteria, and other biological entities to produce nanoparticles (El-Esawy et al., 2024, 2025; Khalifa et al., 2024). Fungi are particularly advantageous candidates for serving as microbial cell factories for nanoparticle production. Their capacity to secrete large amounts of extracellular redox enzymes allows for the efficient reduction and stabilization of metal ions, simplifying downstream processing compared to intracellular synthesis [ 25 , 26 ]. While various fungi have been extensively explored for nanoparticle biosynthesis, the genus Lecanicillium (Ascomycetes, Hypocreales) presents a unique, underexplored opportunity. Known primarily as an entomopathogenic and mycoparasitic, possess robust metabolic machinery capable of secreting diverse bioactive metabolites agent [ 27 – 29 ]. Although Lecanicillium lecanii has been reported to synthesize silver nanoparticles [ 30 ], the specific potential of Lecanicillium aphanocladii for fabricating metal oxide nanoparticles remains uninvestigated. To the best of our knowledge, this is the first study to investigate L. aphanocladii AUMC-16936 as a fungal bio-factory for the biosynthesis of CuONPs. This research aims to characterize the mycosynthesized CuONPs produced by this novel bio-source, as well as validate their biological functionality by assessing their efficacy in inducing physiological, biochemical, and molecular defense responses in potato plants against Fusarium wilt. This study bridges the gap between microbial biotechnology and agriculture, offering a dual solution: the green manufacturing of nanomaterials via fungal metabolism and their subsequent application as sustainable crop protection agents. Materials and methods Reagents and Materials Copper sulphate pentahydrate (CuSO4.5H2O) (99.5%), were purchased from Sigma-Aldrich, Germany. Potato dextrose agar (PDA) and potato dextrose broth (PDB) were purchased from Oxoid, England. Isolation of Lecanicillium aphanocladii The fungus was identified from certain contaminated produced PDA plates that were stored in the lab at room temperature, or around 26°C ± 2. After that, it was sub-cultured in aseptic conditions on fresh PDA plates and incubated for seven days at 26±°C. Following incubation, fungal growth was preserved in glycerol at -80°C for further use after the culture purity was evaluated. Conventional morphological criteria were used to identify the selected fungus. The colony's density and aerial mycelium were observed. As previously reported by [ 31 ], micro morphological characteristics were investigated using a light microscope (Olympus CX51, Japan). Molecular characterization and phylogenetic identification of fungal isolates The selected strain was sent to the Assiut University Mycological Centre (AUMC), Assiut University, Egypt, for extraction of DNA using the Solg™ Genomic DNA Prep Kit (SolGent Co., Ltd, South Korea). PCR was conducted using ITS1(forward) (5′-TCC GTA GGT GAA CCT GCG G -3′), and ITS4 (reverse) (5′- TCC TCC GCT TAT TGA TAT GC -3′). The PCR products were purified with the SolGent PCR Purification Kit-Ultra and sequenced (SolGent Co., Ltd, South Korea). The BlastN technique was used to compare the acquired sequence of this fungus to that of the National Center for Biotechnology Information's (NCBI). For the phylogenetic analysis, sequence alignment was used MegAlign software (version 5.05)[ 32 ]. Biosynthesis and characterization of CuONPs L. aphanocladii mycelial disc (5 mm) was inoculated in 100 mL PDB at 28˚C for 7 days. Then, Whatman filter paper no. 1 was employed for the filtration procedure to yield cell-free filtrates. The filtrate was further purified through centrifugation at 6000 rpm for 5 minutes, at 4 ˚C. The fungal filtrate was examined for the preparation of CuONPs as documented by [ 33 ]. In summary, 30 mL of CuSO 4 ·5H 2 O (0.1 M) was combined with 10 mL of fungal filtrate in a flask, followed by stirring and heating the solution at 90˚C for 5 h. Following the incubation period, the reduction of copper ions was monitored visually and documented as a dark brown, confirming the biosynthesis of CuONPs. The final product was subjected to continuous washing with distilled water, centrifugation at 8000 rpm, and drying in a vacuum oven. The resultant material was thereafter kept at 4 ˚C for investigation. A preliminary validation of CuONP production via mycelial filtrate was evaluated utilizing a UV-visible spectrophotometer (Shimadzu, Kyoto, Japan; Dual Beam Spectrophotometer, UV-1800) over wavelengths from 200 to 800 nm for UV-vis spectroscopy analysis. The investigation utilizing Fourier-transform infrared (FTIR) spectroscopy was conducted with a Perkin-Elmer 1430 infrared spectrophotometer, USA; that features an IR Affinity-1 model. It has a resolution of 4 cm − 1 and a wavelength ranges from 400 to 4000 cm − 1 . Prior to examination, the powdered CuONPs were dehydrated and subsequently combined with KBr to create pellets for examination [ 34 ]. FTIR spectroscopy measures infrared radiation at certain wavelengths, revealing functional group interactions during reduction. In addition, the morphological properties of CuONPs were analyzed using transmission electron microscopy (TEM). A limited quantity of nanoparticle dispersion was deposited onto a carbon-coated copper grid to produce thin films of CuONPs. The samples underwent drying in a vacuum desiccator and were subsequently analyzed using a TEM microscope (JEOL JEM 1400; Japan). After that, X-ray diffractometer (Bruker Co.D8 Discover, Cu target, Wavelength 1.54A, 40 Kv 40 mA, Germany) were implemented. In vitro evaluation of the antifungal efficacy of CuONPs against F. oxysporum F. oxysporum f. sp. lycopersici RCMB008001 was obtained from the Mycology Regional Center, Al-Azhar University, Cairo, Egypt. The pathogenicity test was then confirmed according to [ 35 ]. Various concentrations of CuONPs (25, 50, and 100 ppm), and Maxim XL fungicide used as positive control (3.5%) were assessed for their impact on the radial growth of F. oxysporum , following the methodology established by [ 36 ]. A 0.5 cm diameter disc containing actively growing mycelium from a 7-day-old fungal culture, was positioned at the center of each agar plate. The plates were incubated at 28°C ± 2 for a duration of 4 days. F. oxysporum growth inhibition percentage was obtained using the subsequent equation: Inhibition of pathogen proliferation (%) is calculated using the formula: (control growth − treated growth) / (control growth) × 100. Preparation of fungal inoculum To evaluate the efficacy of CuONPs over Fusarium root rot, a spore suspension of F. oxysporum was grown on PDB [ 37 ]. The spore suspension was agitated and cultured at 160 rpm and 30°C until the media became turbid. The spore solution was further diluted with sterilized distilled water to achieve the target spore concentration of 1 X 106 conidia/ mL. In vivo experiments Analysis of the antifungal properties of CuONPs in combat to Fusarium root rot Effectiveness of CuONPs in mitigating Fusarium rot induced by F. oxysporum was evaluated utilizing potato cultivar ( Solanum tuberosum L.) called Caruso, originating from Germany and imported by Daltex Co., 42 Wadi Al Nile, Gazirat Mit Oqbah, Agouza, Giza Governorate, Egypt. Each treatment was replicated three times, with four to five plants per replicate, using 45 cm diameter plastic pots containing 12 kg of soil (clay: sand, 3:1 v/v), which underwent autoclaving twice for 30 min at 121°C. The physicochemical properties of the soil were as follows: P = 2.4, N = 0.7, K = 34.3, Mg = 23.2, Na = 6.1, Ca = 5.6, and Cl = 6.69 mg/kg. The soil exhibited alkalinity (7.1) and electrical conductivity (3.5 mS/cm). The pots were irrigated daily with tap water until full germination occurred and 500 ml of a fungal spore suspension containing 1 × 106 conidia/mL. On the seventh day after sowing, germinated seedlings were divided into three primary infected groups: a control (tap water), spraying (100 ml), watering (100 ml) and infected group (500 ml of a fungal spore suspension containing 1 × 106 conidia/mL for one week). The infected group was further subdivided into three treatment groups that received infected group, foliar spraying (100 ml) and watering (100 ml) weekly with CuONPs (100 ppm). The experiment was arranged in a completely randomized design with three replicates. Plants were harvested after 60 days of growth for subsequent analysis. The shoot length, root length, fresh weight and dry weight were measured for estimating the potato growth performance. The weight of biomass (g) was ascertained by drying it in a vacuum oven (60°C/3 days). Physiological studies Estimation of hydrogen peroxide (H 2 O 2 ) [ 38 ] identified hydrogen peroxide absorbance at 390 nm, with an extinction coefficient of 0.28 µM –1 cm –1 . Leaf tissue weighing 200 mg was homogenized for 10 minutes in an ice bath with 1 mL of an extraction mixture comprising 1 M potassium iodide, 0.1% (w/v) trichloroacetic acid, and 10 mM potassium phosphate buffer at pH 5.7. Results were expressed as micromoles per gram of fresh weight. Estimation of malondialdehyde content (MDA) The thiobarbituric acid (TBA) assay quantified malondialdehyde (MDA), a lipid peroxidation marker, at wavelengths of 532 and 600 nm, as described by [ 39 ]. Homogenize 0.5 g of freshly harvested leaves in 10 ml of 5% (w/v) TCA. The homogenate underwent centrifugation for 15 minutes at 4000 rpm. TBA at a concentration of 0.677% (w/v) was mixed with equal volumes of supernatant. Following a 15-minute exposure to a boiling water bath, the mixture rapidly cooled down for 10 minutes in cold water. Assessment of various non-enzymatic and enzymatic antioxidants Assessment of total antioxidant capacity (TAC) The phosphomolybdenum method was employed to quantify total antioxidant capacity (TAC), with the sample's absorbance measured at 765 nm following cooling. The TAC reagent was prepared using sulfuric acid (0.6 M), ammonium molybdate tetrahydrate (4 mM), and sodium phosphate dibasic solution (28 mM) as described by Prieto et al. (1999). The ethanolic extract was mixed with TAC and heated for 90 minutes. Assessment of ascorbic acid content (ASA) [ 41 ]quantified the concentration of ascorbic acid (ASA) in leaf tissue using a 5% sulfosalicylic acid solution for extraction. The reaction includes leaf extract, 2% sodium molybdate, 0.15 N sulfuric acid, and 1.5 mM disodium hydrogen phosphate. Following a 45-minute exposure to a 60°C water bath, the mixture was subsequently cooled and subjected to centrifugation. Absorbance was measured at 660 nm. ASA was quantified in mg/g dry weight through the application of a calibration curve. Assessment of reduced glutathione level (GSH) [ 42 ], revealed a reduction in GSH levels. A quantity of 0.1 g of freshly harvested leaves was homogenized in 5 ml of 3% w/v sulfosalicylic acid and subsequently centrifuged for 10 minutes at 10,000 rpm. In the reaction mixture, 0.5 ml of 0.5 mM potassium phosphate buffer (pH 7.0), 0.5 ml of tissue extract, and 50 µl of 3 mM DTNB (5,5′-Dithiobis(2-nitrobenzoic acid) were utilized. Appropriate quantities were utilized. Absorbance was measured at 412 nm using the GSH standard curve to estimate GSH concentration. Assessment of total phenolic content The total phenolic content was calculated by measuring absorbance at 650 nm and comparing it to a standard curve derived from various gallic acid concentrations, as outlined by [ 43 ]. Following the extraction of 0.1 g of desiccated tissues in 10 mL of 95% ethanol, the supernatants were combined and adjusted to a final volume of 10 mL. Following the combination of 1 mL of the extract with 0.1 mL of Foline reagent and 1 mL of Na 2 CO 3 , the resulting mixture was diluted to a final volume of 5 mL using distilled water. Assessment of peroxidase (POD), ascorbate peroxidase (APX), polyphenol oxidase (PPO) activity The extraction process for the antioxidant enzyme matched to the guidelines established by [ 44 ]. The technique employed to assess POD was that of [ 45 ]. The activity of POD was noted at 470 nm, and calculated using a coefficient of extinction of 26.6 mM⁻¹ cm⁻¹. The activity of APX was quantified utilizing the method established by [ 46 ]. The absorbance at 290 nm diminished during the hydrogen peroxide-mediated oxidation of ascorbate. The procedure for quantifying PPO was delineated by [ 47 ]. The enzyme activity was quantified as µM/g f.wt. min − 1 . Then, the absorbance was recorded at 420 nm utilizing an extinction coefficient of 26.40 M − 1 cm − 1 . Evaluation of gene expression by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and RNA extraction. RNA from potato leaves was extracted utilizing the Qiagen RNase Mini Kit. A thermocycler (MJ Research, Inc., PTC-100TM Programmable Thermal Controller, USA) was utilized to synthesize 20 µl of complementary DNA (cDNA) by RNA reverse transcription. Following one hour of enzyme activation at 42°C, a five-minute inactivation phase at 95°C ensued. Conduct triplicate qRT-PCR utilizing Fermentas SYBR Green PCR Master Mix (USA). In each reaction, a 25 µl mixture of primer pairs ( CHI , PIN2 , IbBBx24 , and PR-1b ) as listed in Table 1 was employed. The reaction was conducted using the RotorGene 6000 (QIAGEN, ABI System, USA). The study utilized the ACTIN gene as a reference. The genes analyzed had their relative expression quantified and calculated utilizing the method of [ 49 ]. Table 1 Particular primers utilized in this research Gene name Abbreviation Forward (F) and reverse (R) primer Reference gene ACTIN F: 5′-GGTAACATTGTGCTCAGTGGTGG-3′ R: 5′-AACGACCTTAATCTTCATGCTGC-3′ Pathogenesis related protein PR-1b F: 5′-GGCATCCCGAGCACAAAAT-3′ R: 5′-CTGCACCGGAATGAATCAAGT-3′ Box-transcription factor IbBBx24 F: 5′-AAACGCACAATTGGAGCCAC-3′ R: 5′-GTAGGTCATCAACGGCCCAA-3′ Protease inhibitor PIN2 F: 5′-ATGAGCCCAAGGCAAATATGTAC-3′ R: 5′-GCCAATCCAGAAGATGGACAA-3′ Chalcone isomerase CHI F: 5′-TGGTGGCCTAGACAACGATGAGTT-3′ R: TCACACTCCCAACTTGGTTTCCCT-3′ Statistical analysis The findings were reported as the mean of three replicates, and the standard error (SEs) was computed. Two-way ANOVA was employed for statistical analysis to identify significant differences among treatments. Analyses were conducted using XLSTAT software (version 2014.5.03), with a significant threshold was set at p ≤ 0.01. Results Morphological and molecular characterization and phylogenetic identification of L. aphanocladii isolate Macroscopic characteristics revealed that they were white in color, high on the medium's surface, and had a reddish appearance at the bottom of the plates. Additionally, the microscopic structures of L. aphanocladii revealed that the fungus produced conidia directly on the hypha and possessed cylindrical aphanophialides that carried phialo-conidium (Fig. 1 A). The sequences of L. aphanocladii AUMC-16936 strain and deposited in Gene bank with accession No.PV549634 showed 99.14% − 100% similarity and 93% − 100% coverage with several strains of the same species, Trichoderma asperellum , included as outgroup strain (Fig. 1 B). L. aphanocladii AUMC-16936 was deposited in the DDBJ GeneBank nucleotides sequence database with accession number (PV549634). Mycosynthesis and characterization of CuONPs In this study, CuONPs were successfully biosynthesized using the fungal filtrate of L. aphanocladii . The fungal filtrate shifted from a yellowish to a dark brown color, signifying the biofabrication of nanoparticles. Subsequently, various characterization techniques were employed to verify the production of CuONPs. The characteristic peak of CuONPs was observed at 350 nm using UV-visible spectroscopic analysis, as illustrated in Fig. (2). Additionally, nanoparticles were examined using TEM to reveal further morphological characteristics. The TEM analysis, as illustrated in Fig. (3A), demonstrated the existence of rod-shaped particles with sizes varying from 15.24 ± 4.19 nm. The sharp XRD peaks confirm the crystalline nature of the mycosynthesized CuONPs (Fig. 3 B). The narrow peak widths and high intensities indicate small particle size. Prominent diffraction peaks at 2θ = 19.113°, 29.786°, 32.596°, 35.577°, 38.729°, 48.783°, 53.556°, 58.210°, 61.549°, 66.080°, and 68.052° correspond to the (110), (111), (111), (002), (202), (113), (020), (202), (311), (220), and (311) planes of monoclinic CuONPs (JCPDS card no. 01-007-2551). Additionally, Fig. (3C) displays the FTIR spectra of the cell-free filtrate and the produced nanoparticles within the 400–4000 cm − 1 range. The data obtained unequivocally demonstrated the emergence of a new band at 422 cm − 1 in the spectrum of the produced nanoparticles, indicative of CuO nanoparticles. Nevertheless, there are no bands corresponding to any of these bands in the spectrum of the cell-free filtrate. The recorded results indicated that both spectra (for cell-free culture and created nanoparticles) displayed principal bands at 3432, 2932, 1638, 1372, and 1041 cm − 1 , demonstrating the involvement of the cell-free L. aphanocladii filtrate in nanoparticle production. extract. In vitro antifungal efficacy of CuONPs The antifungal activity of CuONPs was evaluated at three concentrations on PDA using the radial growth inhibition (RGI) assay. The antifungal activity of CuONPs exhibited higher effect than Maxim 4FS as shown in Fig. 4 A, and B. The radial growth of F. oxysporum strain was recorded as 49 ± 2.0, 26 ± 0.5, and 20.3 ± 0.3 mm, respectively, at concentrations of 25, 50, and 100 ppm in CuONPs supplemented media. The percentages of F. oxysporum growth suppression were recorded at 39%, 68%, and 75%, respectively (Fig. 4 C). In vivo impact of CuONPs on growth parameters of potato plants Impact of CuONPs on plant height, fresh, and dry masses This study assessed the efficacy of CuONPs in controlling soil-borne Fusarium wilt disease in potatoes by a pot bioassay. Three months post-seeding, the impact of CuONPs on potato was assessed regarding plant lengths, as well as fresh and dry weights (Fig. 5 , 6 ). The findings demonstrate a notable enhancement in plant growth metrics following the exposure of potato plants to 100 ppm of CuONPs, administered either via spraying (SN) or watering (WN), resulting in increases of 15% and 10%, respectively, compared to the control treatment. In terms of fresh and dry biomasses, potato plants treated with CuONPs exhibited a notable enhancement, with increases of 55% and 113% in fresh weight, respectively, and 67% and 150% in dry weight, respectively, as compared to the negative control in healthy plants (Fig. 6 B, C). Impact of CuONPs on oxidative stress Figure (7) illustrates that a concentration of 100 ppm of CuONPs did not induce oxidative stress in potato plants, as there was no significant elevation in H 2 O 2 and MDA levels in the CuONP-treated plants compared to the negative control. Conversely, potato plants infected with F. oxysporum exhibited a significant 29% and 51.8%increase in H 2 O 2 and MDA content, respectively, relative to the negative control group. Furthermore, treatment with CuONPs (SN and WN) effectively mitigated the oxidative stress caused by Fusarium infection, resulting in a reduction of H 2 O 2 and MDA levels by 14% and 15%, 38.7 and 34.5% respectively, compared to the infected potato plants. Impact of CuONPs on antioxidant compounds The current investigation demonstrated a considerable increase in total antioxidant capacity (TAC) in Fusarium-infected plants following the administration of CuONPs, with enhancements of 68% and 48% observed through spraying and watering, respectively, compared to infected plants alone. Conversely, infection with F. oxysporum resulted in a substantial reduction in total antioxidant capacity (TAC) by 30% relative to uninfected potato plants (Fig. 8 A). The application of CuONPs, both singly (SN and WN), resulted in a substantial increase in total phenolic (TP) content by 88% and 60%, respectively, as compared to the negative control plants. A notable enhancement in TP content of infected plants treated with CuONPs was recorded, with increases of 194% and 145% attributed to the application of SN and WN, respectively (Fig. 8 B). The utilization of SN or WN markedly increased the levels of GSH and ASA in 60-day-old diseased potato plants. The enhancement ratios were 393% and 345% for GSH, and 39% and 34% for ASA, in comparison to infected plants. Infection with F. oxysporum led to a substantial reduction in the levels of GSH and ASA in 60-day-old potato plants, decreasing by 71% and 21%, respectively, compared to the control group (Fig. 8 C, D). Impact of CuONPs on enzymatic activity Figure 9 illustrates that infection with F. oxysporum reduced the activities of POD, PPO, and APX in 60-day-old potato plants by 28%, 7%, and 75%, respectively, in comparison to healthy plants. In contrast, CuONPs administered via either SN or WN markedly improved the enzyme levels, approaching those observed in healthy plants. The maximum value was seen at APX enzyme, with CuONPs considerably enhancing enzyme activity by 825% and 600% for spraying and watering, respectively, in comparison to infected plants. Moreover, the utilization of SN or WN markedly enhanced the activity of POD and PPO in infected plants by 40% and 29% for POD, and 22% and 19% for PPO, respectively. Influence of CuONPs on the relative gene expression levels of PR1b, PIN2, IbBBx24 , and CHI As illustrated in Figure (10), potato plants infected with Fusarium and treated with CuONPs (SN and WN) exhibited elevated levels of PR1b, IbBBx24 , and CHI in comparison to other treatments. Nonetheless, PIN2 in infected plants exhibited a significant increase when compared to other treatments. Interestingly, the expression of the PIN2 gene was not affected by CuONPs treatments, whether applied through foliar techniques or watering. Pearson Correlation Coefficient Pearson's simple correlation provided a comprehensive visualization of the interrelationships among physiological, biochemical traits and molecular approaches in plants observed in various treatments with mycosynthesized CuONPs applications (Fig. 11 ). The findings revealed strong positive correlations between growth parameters, in addition, phenolic content, and TAC. Growth parameters were also found to be positively related with augmented enzymatic antioxidants (PPO, POD, and, APX). Furthermore, the expression levels of PR1b, IbBBx24 , and CHI showed positive correlations with (PPO, POD, and, APX), phenolic content, ASA, and GSH. Conversely, oxidative stress markers (H₂O₂, MDA) were showed strong negative correlations with growth parameters and antioxidants, indicating that increased oxidative damage is associated with reduced plant growth and metabolic activity. The positive correlation between H₂O₂ and PIN2 suggests that hydrogen peroxide may act as a signaling molecule inducing stress-related gene expression. Overall, the heat map not only visualizes the complex interplay between growth, antioxidants, and stress markers but also provides a foundation for understanding how plants balance growth and stress responses at the biochemical level. Such insights are crucial for developing strategies to enhance plant resilience in challenging environments. Principal component analysis (PCA) In comparison to the CuONPs treatments, the PCA results linked the gene expression data with physiological factors (Fig. 11 ). According to the study's findings, 87.11% of the variation was explained by the first four PCs with eigenvalues greater than 1. PC1 was the most significant, accounting for 65.21% of the overall variation. GSH, ASA, phenolic, PPO, APX, and CHI were the metrics that showed the strongest connection with the PC1, in decreasing order. PC2, which was responsible for 21.90% of the overall change, was most impacted by Ibbx24, PR-1b , fresh weight, dry weight, POD, shoot length, and TAC, in decreasing order. Significant correlations were found between PC3, which explained 12.89% of the variation, and adverse impact factors, MDA, PIN2 , and H 2 O 2 . Additionally, CUONPs were found to favour growth, physiological characteristics, and gene expression, according to PCA. Nonetheless, there was a strong correlation between the contaminated potato and MDA, PIN2 , and H 2 O 2 . Discussion The search for sustainable bio-manufacturing platforms has positioned fungi as promising candidates for the green synthesis of nanomaterials. In this context, our study successfully demonstrates, for the first time, the metabolic potential of Lecanicillium aphanocladii AUMC 16936 to function as a microbial cell factory for the production of CuONPs, and establishes their potent efficacy against Fusarium wilt in potato plants. The biosynthetic process was visually indicated by a distinct color change of the reaction mixture from pale yellow to dark brown hue, indicating its role as a reducing and capping agent that converts copper sulfate into CuONPs and stabilizes them in colloidal sate. Biologically active metabolites derived from the fungal extract function as a capping agent, inhibiting nanoparticle agglomeration and modifying their biological activity [ 50 , 51 ]. The analysis of the UV-visible spectra revealed an obvious absorption peak at around 350 nm, indicating the successful production of CuONPs by the tested fungus. The peak of CuO's UV absorption typically exhibits an absorption range between 280 nm and 360 nm [ 52 ]. Moreover, the absorption peak of excited CuONPs seen at 350 nm signifies a monodispersed sizes are distributed within the overall mixture [ 53 ]. In order to gain additional understanding of the structural characteristics of the mycosynthesized CuONPs, the particles were examined using TEM. The nanostructured particles displayed a rod-shape with diameters ranging from 15.24 ± 4.19 nm. This outcome parallels findings in earlier studies regarding the shape of CuNPs and CuONPs [ 54 , 55 ]. Furthermore, the monoclinic structure of the mycosynthesized CuONPs was indicated by the XRD analysis, which showed diffraction peaks that matched the standard CuO. FTIR spectroscopy analysis was conducted to validate the production of nanoparticles and the examination of their interactions the produced nanoparticles and active metabolites from the cell-free filtrate of L. aphanocladii . The results clearly demonstrated the effective synthesis of CuONPs. The distinctive peak of CuO was detected at 422 cm⁻¹. These findings align with numerous investigations that documented peaks at wavenumbers between 500–700 cm –1 , indicating the production of CuO [ 56 , 57 ]. The data indicated that the prominent broad absorption band between 2500 and 3750 cm − 1 corresponds to the O–H stretching of hydroxyl groups in the compound, while the peak at 2095.94 cm − 1 is attributed to the alkene (C = C), and the peak at 1638.94 cm − 1 may be ascribed to the amine group. During The peak at 1554.75 results from the C = C stretching of the aromatic group in tertiary amides, tannins, saponins, flavonoids, and terpenoids. The peak at 1372.40 cm − 1 may correspond to C-N stretching in aliphatic amines, while the peak at 1041.47 cm − 1 is related with C-O stretching in phenols, alcohols, esters, or, ethers and may also indicate C-N stretching in aromatic amines [ 58 , 59 ]. The existence of several functional groups in the biomass of fungal filtrate indicates their significance in the reduction of metal precursors to produce CuONPs, then capped to enhance their stability. Numerous widely available antifungal had minimal efficacy towards Fusarium spp. Consequently, significant focus is necessary to develop a biodegradable and efficient antifungal agent against F. oxysporum [ 60 ]. The antifungal effectiveness of CuONPs was estimated at three doses on PDA using the radial growth inhibition (RGI) assay, recognized as an effective and dependable approach for assessing the fungicidal properties of nanomaterials [ 61 ]. Mechanistically, CuONPs demonstrate antifungal properties by generating reactive oxygen species (ROS) via Haber–Weiss and Fenton-like processes, which compromise fungal cell walls by oxidizing chitin and glucan [ 62 ]. They also hinder respiratory metabolism and downregulate chitin synthase genes, resulting in morphological alterations. Furthermore, Cu + and OH – ions engage with microbial proteins, resulting in the impairment of DNA, cell membranes, proteins, and lipids, finally leading to cellular death [ 63 , 64 ]. This potent direct toxicity suggests that L. aphanocladii -derived CuONPs are viable candidates for replacing synthetic fungicides. Consequently, to validate the agricultural utility of this bio-product, in vivo trials revealed that application method plays a critical role in efficacy. Three months post-seeding, the impact of CuONPs on the growth parameters of potato was assessed. The findings demonstrate a notable enhancement in plant growth measurements following the exposure of potato to 100 ppm of CuONPs, administered either through spraying (SN) or watering (WN), compared to the control treatment. Our findings align with those of other nanoparticles, indicating that NPs enhance overall plant growth [ 58 , 65 ]. Thus, these results suggest that CuONPs can be utilized to promote plant growth and development. Furthermore, the application of CuONPs to infected plants (by spraying and watering) had a substantial stimulatory effect on the height of potato plants, as well as on their fresh and dry biomass, compared to the control group. It is suggested that, this increase in fresh weight may arise from the nanoparticles enhancing the functionality of photosystems I and II, as well as the redox state of plastoquinone within the electron transport chain [ 66 ]. Oxidative stress induced by F. oxysporum infection caused significant damage to plant cells, resulting in elevated levels of MDA and H 2 O 2 in the potato plants compared with those in the healthy control. These findings are in line with those of [ 67 ], who demonstrated that increasing MDA and H 2 O 2 levels in various potato varieties illustrate the impact of Fusarium wilt disease. Moreover, the results demonstrated that both CuONPs (SN and WN) displayed enhanced H 2 O 2 scavenger activity, and MDA content, compared to the untreated control. Given the significance of copper as a microelement for plants, copper nanoparticles may therefore trigger defense systems in response to pathogen inoculation by producing extra proteins to stop pathogen entry or subsequent dissemination [ 58 ]. These results are similar to those of [ 68 ], who reported that the application of copper nanoparticles under stress conditions resulted to decreasing of MDA and H 2 O 2 . Fusarium is a mycotoxins producing fungus, which induce ROS generation at the cellular level, potentially resulting in oxidative damage in plants [ 69 ]. To mitigate oxidative stress and defend against its detrimental effects, plant cells initiate antioxidant systems, encompassing the synthesis of both non-enzymatic and enzymatic substances [ 70 ]. The current investigation revealed that the treatments of CuONPs with F. oxysporum enhanced the levels of secondary metabolites, including ascorbic acid, glutathione, total antioxidants, and total phenolic compounds, in comparison to infected plants. ASA and GSH molecules have a crucial antioxidant function and participate in various cellular signals within plant stress response pathways, including photosynthesis and hormone biosynthesis [ 27 , 71 , 72 ]. Furthermore, Flavonoids and phenols are secondary metabolites, and their elevated levels are associated with the enhancement of induced resistance in plants, encompassing phytoalexins. They participate in hypersensitive responses, cell wall lignification, cellular apoptosis, and contribute to disease resistance [ 73 , 74 ]. Our findings align with those of Diana et al. [ 75 ], who demonstrated the efficacy of Graphene-Cu nanocomposite in enhancing resistance and eradicating F. oxysporum infection in tomato plants by elevating levels of GSH, flavonoids, and other antioxidant enzymes. The antioxidant response in plants comprises many enzymatic molecules that remove reactive oxygen species (ROS) [ 76 ]. Peroxidases (APX, POD, and PPO) are recognized for their roles in regulating plant development, offering defense against diverse stresses, and facilitating the production of lignin; they bolster plant defense against diverse pathogens via strengthening cell wall barriers and communicating signals to adjacent healthy cells [ 77 ]. The results are consistent with those reported here, indicating that potato plants exhibit enhanced stress tolerance under these treatments. The application of CuONPs, whether through spraying or watering, markedly enhanced the activity of POD, PPO, and APX in 60-day-old infected potato plants relative to the infected controls. Similarly, our findings are corroborated by the research of Ashraf et al. [ 65 ], which indicated that application of AgNPs enhanced phenolics contents and antioxidant enzyme activity(PAL, POD, and PPO) in tomato plants infected with F. oxysporum . The preventive impact of CuONPs against Fusarium wilt disease in potato plants by enhancing both enzymatic and non-enzymatic defense mechanisms. The induction of this systemic resistance was further confirmed at the molecular level. The obtained results stated that the expression of the tested genes ( PR1b , IbBBx24 , and CHI) upregulated significantly in response to CuONPs treatments, suggesting an increase in resistance mechanism of potato plants. The upregulation of pathogenesis related protein (PR1b), and chalcone isomerase ( CHI ) triggers the salicylic acid-signaling pathway, and catalyzes the formation of flavonoids, respectively [ 78 , 79 ]. This could be considered a systemic acquired resistance response. Moreover, B-box (BBX) family transcription factor IbBBX24 modulates the jasmonic acid (JA) pathway in sweet potato. IbBBX24 is crucial for the regulation of jasmonic acid production and signaling, enhancing resistance to Fusarium wilt and improving yield in sweet potato. Our results are in similar line with several reports addressed the role of these genes in enhancing pathogen resistance plants [ 78 , 80 ]. Interestingly, PIN2 gene expression was upregulated significantly in infected plants with F. oxysporum only. Moreover, there was no difference in the expression of this gene in infected plants treated with CuONPs compared to healthy plants. This might be due to the antifungal effect of CuONPs inside plant tissues, which led to normal expression rate of PIN2 gene compared to infected control plants. It is noteworthy that the mode of application significantly influenced efficacy, with foliar spraying consistently outperforming soil watering. This may be attributed to the fact that foliar-applied nanoparticles primarily penetrate the leaves through stomata and are subsequently conveyed through various plant tissues using symplastic and apoplastic pathways[ 81 , 82 ]. Nutrients applied to the leaves are moved downwards and distributed to other plant parts. Likewise, the application of NPs on the leaves resulted in a higher NPs content within the root system, aiding nutrient absorption by root cells and enhancing their resistance to disease[ 83 ]. Thus, it can be posited that foliar application of nanoparticles augments the efficacy of plant protection strategies in comparison to conventional soil-root methods. Therefore, L. aphanocladii AUMC-16936 serves as an efficient and eco-friendly microbial cell factory for the production of bioactive CuONPs inhibiting F. oxysporum growth while also stimulating the potato plant's immune system via genetic and antioxidant pathways. Conclusions In conclusion, this study provides the first evidence utilizing L. aphanocladii AUMC-16936 as a novel microbial cell factory for the green biosynthesis of CuONPs. The fungal bio-manufacturing process yielded highly stable, protein-capped nanorods. with dual-functional capabilities: direct fungicidal activity against F. oxysporum and the ability to prime molecular defense mechanisms in potato plants. Foliar application of these biogenic nanoparticles proved to be the superior delivery strategy, significantly reducing disease severity and enhancing crop yield by upregulating key antioxidant and defense gene networks. By transforming a fungal metabolite stream into a high-value nano-agrochemical, this work bridges the gap between industrial biotechnology and sustainable agriculture. Future research should focus on optimizing fermentation conditions to scale up this mycosynthesis process, while also assessing their long-term environmental impacts such as soil accumulation and effects on non-target organisms. Declarations Consent for publication All authors approved the manuscript. Competing interests The authors declare no competing interests. Funding: This research was funded by Princess Nourah bint Abdulrahman University Researchers Supporting Project number (PNURSP2026R318), Princess Nourah bint Abdulrahman University, Riyadh, Saudi Arabia. Author Contribution Conceptualization: N.A.E, M.A.E, and D.E.E.; methodology: N.A.E, M.A.E, D.E.E. and M.H.; formal analysis and software: D.E.E. and M.H.; investigation: N.A.E, M.A.E, D.E.E. and M.H.; writing—original draft preparation: D.E.E. and M.H; writing—reviewing and editing: D.E.E., F.A.S., and M.H. All authors have read and agreed to the published version of the manuscript. 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09:23:10","extension":"png","order_by":63,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":24897,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-8360033/v1/c26a50e28e4160f73faeb2ab.png"},{"id":98779265,"identity":"20da5c86-9536-46e6-a723-dec8823f2f58","added_by":"auto","created_at":"2025-12-22 12:30:07","extension":"xml","order_by":64,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":184089,"visible":true,"origin":"","legend":"","description":"","filename":"5515f30a83d241ba9c58148ac0f3b56f1structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8360033/v1/f98f36f3d6e9e96f0d0e4326.xml"},{"id":98753804,"identity":"13b09ddc-e16c-4bf7-ae3e-f847ea812887","added_by":"auto","created_at":"2025-12-22 09:23:11","extension":"html","order_by":65,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":208750,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8360033/v1/8fb31fa3f5ea29e47f1006ab.html"},{"id":98777936,"identity":"fc39c4f6-d57c-40d1-b3bc-bb1dec5fa269","added_by":"auto","created_at":"2025-12-22 12:28:41","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":365208,"visible":true,"origin":"","legend":"\u003cp\u003eA. Morphological characterization and B. phylogenetic tree of \u003cem\u003eLecanicillium aphanocladii \u003c/em\u003eAUMC 16936.\u003c/p\u003e","description":"","filename":"fig.11.png","url":"https://assets-eu.researchsquare.com/files/rs-8360033/v1/c4db49d88d4c101056fbb1e3.png"},{"id":98779037,"identity":"394b25f3-a9f6-449f-8815-0815cc4992cc","added_by":"auto","created_at":"2025-12-22 12:29:53","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":73454,"visible":true,"origin":"","legend":"\u003cp\u003eUV–Vis spectrum of mycosynthesized CuONPs by \u003cem\u003eLecanicillium aphanocladii \u003c/em\u003ecell free\u003c/p\u003e\n\u003cp\u003eextract.\u003c/p\u003e","description":"","filename":"fig.21.png","url":"https://assets-eu.researchsquare.com/files/rs-8360033/v1/2933c2326c69e55d43c1c615.png"},{"id":98778237,"identity":"6c9e567c-0596-449b-9ae4-3a8ac0c36220","added_by":"auto","created_at":"2025-12-22 12:29:02","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":564977,"visible":true,"origin":"","legend":"\u003cp\u003ePhysiochemical characterization of CuONPs from \u003cem\u003eLecanicillium aphanocladii\u003c/em\u003e. (A)Transmission electron image at magnification 100 nm. (B) X-ray diffractometer (C)The FTIR of fungal filtrate (1) versus biosynthesized CuONPs (2) \u003cem\u003eLecanicillium aphanocladii\u003c/em\u003e cell free extract.\u003c/p\u003e","description":"","filename":"fig.31.png","url":"https://assets-eu.researchsquare.com/files/rs-8360033/v1/3476cb13e331d4848fe6391c.png"},{"id":98780356,"identity":"4ade10ff-bc0f-47b7-b0c0-1f60d62abb21","added_by":"auto","created_at":"2025-12-22 12:31:14","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":382289,"visible":true,"origin":"","legend":"\u003cp\u003eRadial growth of \u003cem\u003eF. oxysporum \u003c/em\u003eat varying doses (ppm)\u003cem\u003e \u003c/em\u003eof CuONPs \u003cstrong\u003e(A)\u003c/strong\u003e.\u003cem\u003e \u003c/em\u003eGrowth diameters at different doses of CuONPs (\u003cstrong\u003eB\u003c/strong\u003e); growth inhibition\u003cem\u003e \u003c/em\u003epercentages (\u003cstrong\u003eC\u003c/strong\u003e). Letters on mean values statistically significant differences between the mean of the represented control and the treatments at the same time (p ≤ 0.01)\u003c/p\u003e","description":"","filename":"fig.41.png","url":"https://assets-eu.researchsquare.com/files/rs-8360033/v1/403778fa19d1c604203efb4e.png"},{"id":98753772,"identity":"c23d21b9-4aac-4684-8390-a1de6691b7cf","added_by":"auto","created_at":"2025-12-22 09:23:10","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":317337,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of CuONPs (100 ppm) on potato plants cultivated in pot trials infected with \u003cem\u003eF. oxysporum\u003c/em\u003e, in comparison to control sets.\u003c/p\u003e","description":"","filename":"fig.51.png","url":"https://assets-eu.researchsquare.com/files/rs-8360033/v1/f0c2c55bef7062005e69923b.png"},{"id":98753754,"identity":"967f5ec1-05d0-4417-8046-6da8882b2e6c","added_by":"auto","created_at":"2025-12-22 09:23:09","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":58421,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of foliar spray (SN) and watering (WN) with CuONPs (100 ppm) on (A); shoot length, (B); fresh weight, and (C); dry weight of potato infected (Inf) with \u003cem\u003eF. oxysporum\u003c/em\u003e compared to control. Letters on mean values statistically significant differences between the mean of the represented control and the treatments at the same time (p ≤ 0.01)\u003c/p\u003e","description":"","filename":"fig.61.png","url":"https://assets-eu.researchsquare.com/files/rs-8360033/v1/3d03cf7cd37b68ffc10bdd3a.png"},{"id":98753761,"identity":"41ed1102-41c6-47c7-a9f2-e58ef76af541","added_by":"auto","created_at":"2025-12-22 09:23:10","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":40241,"visible":true,"origin":"","legend":"\u003cp\u003eImpact of foliar spray (SN) and watering (WN) with CuONPs (100 ppm) on (A); (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e)\u0026nbsp; and (B) MDA of potato infected (Inf) with \u003cem\u003eF. oxysporum\u003c/em\u003e compared to control. Letters on mean values statistically significant differences between the mean of the represented control and the treatments at the same time (p ≤ 0.01).\u003c/p\u003e","description":"","filename":"fig.71.png","url":"https://assets-eu.researchsquare.com/files/rs-8360033/v1/a80bd72cfb3d582688d468fe.png"},{"id":98778443,"identity":"4e25368f-be58-45ce-930d-8446283a7e2a","added_by":"auto","created_at":"2025-12-22 12:29:15","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":95792,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of foliar spray (SN) and watering (WN) with CuONPs (100 ppm) on (A); total antioxidant capacity (TAC), (B); total phenolic content, (C); ascorbic acid content (ASA), and (D); glutathione content (GSH) of potato infected (Inf) with \u003cem\u003eF. oxysporum\u003c/em\u003ecompared to control. Letters on mean values statistically significant differences between the mean of the represented control and the treatments at the same time (p ≤ 0.01).\u003c/p\u003e","description":"","filename":"fig.81.png","url":"https://assets-eu.researchsquare.com/files/rs-8360033/v1/09b81398df4439fa29cd18cc.png"},{"id":98777610,"identity":"6b851359-d5c3-421e-bfca-f33a800a262c","added_by":"auto","created_at":"2025-12-22 12:28:10","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":54347,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of foliar spray (SN) and watering (WN) with CuONPs (100 ppm) on (A); peroxidase (POD), (B); ascorbate peroxidase activity (APX), and (C); polyphenol oxidase activity (PPO) (µM/g.f.wt.min\u003csup\u003e-1\u003c/sup\u003e) of potato infected (Inf) with \u003cem\u003eF. oxysporum\u003c/em\u003e compared to control. Letters on mean values statistically significant differences between the mean of the represented control and the treatments at the same time (p ≤ 0.01)\u003c/p\u003e","description":"","filename":"fig.91.png","url":"https://assets-eu.researchsquare.com/files/rs-8360033/v1/0daf071a91c79dad67de06ff.png"},{"id":98778411,"identity":"af8f972f-f851-49fe-9e56-f10397742971","added_by":"auto","created_at":"2025-12-22 12:29:14","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":103219,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of foliar spray (SN) and watering (WN) with CuONPs (100 ppm) on the relative gene expression of \u003cem\u003eRP-1b, IbBBx24, PIN2\u003c/em\u003e, and \u003cem\u003eCHI\u003c/em\u003e of potato infected (Inf) with \u003cem\u003eF. oxysporum\u003c/em\u003e compared to control.\u003cstrong\u003e \u003c/strong\u003eLetters on mean values statistically significant differences between the mean of the represented control and the treatments at the same time (p ≤ 0.01).\u003c/p\u003e","description":"","filename":"fig.101.png","url":"https://assets-eu.researchsquare.com/files/rs-8360033/v1/85f719ad49ab124197fee504.png"},{"id":98777946,"identity":"4398acec-4d82-4429-b703-e8200e32a227","added_by":"auto","created_at":"2025-12-22 12:28:42","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":254896,"visible":true,"origin":"","legend":"\u003cp\u003eHeat Map of Pearson Correlation Coefficient analyse the relationships between physiological and molecular characteristics observed in various treatments with CuONPs in plants infected with \u003cem\u003eF. oxysporum\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"fig.111.png","url":"https://assets-eu.researchsquare.com/files/rs-8360033/v1/d8c3ce57e5dfbc3ca46b6ef8.png"},{"id":98753759,"identity":"3c7d5fb8-98dc-44d3-929f-a15c025f3bc0","added_by":"auto","created_at":"2025-12-22 09:23:09","extension":"png","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":44766,"visible":true,"origin":"","legend":"\u003cp\u003ePrincipal Component Analysis (PCA) of potato plant growth, genetic, and physiological parameters at varying concentrations of CuONPs in plants infected with \u003cem\u003eF. oxysporum\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"fig.121.png","url":"https://assets-eu.researchsquare.com/files/rs-8360033/v1/ae9de412fc223eee40cede90.png"},{"id":98777830,"identity":"2adb3eeb-4c8a-4fcd-a12e-8a2f052aa595","added_by":"auto","created_at":"2025-12-22 12:28:32","extension":"png","order_by":13,"title":"Figure 13","display":"","copyAsset":false,"role":"figure","size":375061,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic representation of the effects of CuONPs on various physiological, biochemical parameters, and molecular approach in potato infected with \u003cem\u003eF. oxysporum\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"fig.131.png","url":"https://assets-eu.researchsquare.com/files/rs-8360033/v1/4cf686c9ffef10cd0bb3c1cc.png"},{"id":106809449,"identity":"e3ad254e-ed8b-44d1-a6f7-06b51afd6809","added_by":"auto","created_at":"2026-04-13 16:11:02","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4284634,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8360033/v1/f2ad8eb9-daf6-41e1-bbac-00f360bb52ce.pdf"},{"id":98753756,"identity":"019798c8-8766-489f-8e43-055e38520d9e","added_by":"auto","created_at":"2025-12-22 09:23:09","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":24673,"visible":true,"origin":"","legend":"","description":"","filename":"Table1.docx","url":"https://assets-eu.researchsquare.com/files/rs-8360033/v1/b07d654c08eb8babf83b0c0f.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Exploring the Potential of Lecanicillium aphanocladii AUMC-16936 as a Novel Microbial Cell Factory for the Biosynthesis of CuONPs Against Potato Fusarium Wilt","fulltext":[{"header":"Introduction","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003ePotatoes (\u003cem\u003eSolanum tuberosum\u003c/em\u003e) can significantly contribute to food security by improving availability, access, utilization, and stability [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e].Potato production ranks as the third most significant food crop following wheat and rice, sustaining over 1.3\u0026nbsp;billion individuals [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. However, potato farming encounters considerable obstacles, including fungal infections like \u003cem\u003eFusarium oxysporum\u003c/em\u003e, responsible for Fusarium wilt [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. This soil-borne disease can cause significant yield reductions, jeopardizing food security and farmers' livelihoods. \u003cem\u003eF. oxysporum\u003c/em\u003e is a notable threat because of its ability to infect potato plants over their whole growth cycle, from germination to maturity [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. It infects plants via root systems, injuries, or natural apertures, resulting in vascular wilt, root rot, and ultimately the decline of potato crops [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. The fungus infiltrates the plant's vascular system, resulting in obstructions that hinder the proper water movement and nutrients. Infected plants frequently have dark-brown streaks within the xylem vessels, a characteristic indicative of Fusarium wilt [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. A particularly alarming feature of \u003cem\u003eF. oxysporum\u003c/em\u003e infection is the synthesis of mycotoxins, deleterious secondary metabolites that not only intensify plant damage but also threaten the health of humans and animals upon the consumption of infected tubers [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eExisting control strategies predominantly depend on chemical fungicides, crop rotation, and the utilization of disease-resistant potato cultivars [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. The excessive application of chemical treatments has resulted in the development of resistant fungus strains and environmental pollution [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. The main consequence is that fungicides produce chemical traces on non-target organisms and enter the food chain [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Consequently, these problems highlight the necessity for sustainable, environmentally friendly techniques to effectively address \u003cem\u003eF. oxysporum\u003c/em\u003e infections [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. By tackling the difficulties presented by fungal infections with innovative and sustainable solutions, the agricultural sector can enhance the resilience and production of potato crops, thereby preserving their essential role in global food chains.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003eNanotechnology has arisen as a promising solution in the controlling the spread of agricultural diseases [\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Copper Oxide nanoparticles exhibit promising antifungal capabilities simply because of their diminutive size, enormous surface area, and power to produce reactive oxygen species (ROS). These nanoparticles compromise the integrity of the cell wall and membrane of fungal pathogens, impede enzyme functions, and disrupt metabolic pathways, ultimately resulting in the suppression of infections [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. In green chemistry for nanoparticle synthesis, environmentally benign solvents, non-toxic reducing substances, and safe stabilizers are prioritized. This method utilizes plant extracts, fungi, algae, bacteria, and other biological entities to produce nanoparticles (El-Esawy et al., 2024, 2025; Khalifa et al., 2024).\u003c/p\u003e \u003cp\u003eFungi are particularly advantageous candidates for serving as microbial cell factories for nanoparticle production. Their capacity to secrete large amounts of extracellular redox enzymes allows for the efficient reduction and stabilization of metal ions, simplifying downstream processing compared to intracellular synthesis [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. While various fungi have been extensively explored for nanoparticle biosynthesis, the genus \u003cem\u003eLecanicillium\u003c/em\u003e (Ascomycetes, Hypocreales) presents a unique, underexplored opportunity. Known primarily as an entomopathogenic and mycoparasitic, possess robust metabolic machinery capable of secreting diverse bioactive metabolites agent [\u003cspan additionalcitationids=\"CR28\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Although \u003cem\u003eLecanicillium lecanii\u003c/em\u003e has been reported to synthesize silver nanoparticles [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], the specific potential of \u003cem\u003eLecanicillium aphanocladii\u003c/em\u003e for fabricating metal oxide nanoparticles remains uninvestigated. To the best of our knowledge, this is the first study to investigate \u003cem\u003eL. aphanocladii\u003c/em\u003e AUMC-16936 as a fungal bio-factory for the biosynthesis of CuONPs. This research aims to characterize the mycosynthesized CuONPs produced by this novel bio-source, as well as validate their biological functionality by assessing their efficacy in inducing physiological, biochemical, and molecular defense responses in potato plants against Fusarium wilt. This study bridges the gap between microbial biotechnology and agriculture, offering a dual solution: the green manufacturing of nanomaterials via fungal metabolism and their subsequent application as sustainable crop protection agents.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eReagents and Materials\u003c/h2\u003e \u003cp\u003eCopper sulphate pentahydrate (CuSO4.5H2O) (99.5%), were purchased from Sigma-Aldrich, Germany. Potato dextrose agar (PDA) and potato dextrose broth (PDB) were purchased from Oxoid, England.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIsolation of\u003c/b\u003e \u003cb\u003eLecanicillium aphanocladii\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe fungus was identified from certain contaminated produced PDA plates that were stored in the lab at room temperature, or around 26\u0026deg;C\u0026thinsp;\u0026plusmn;\u0026thinsp;2. After that, it was sub-cultured in aseptic conditions on fresh PDA plates and incubated for seven days at 26\u0026plusmn;\u0026deg;C. Following incubation, fungal growth was preserved in glycerol at -80\u0026deg;C for further use after the culture purity was evaluated. Conventional morphological criteria were used to identify the selected fungus. The colony's density and aerial mycelium were observed. As previously reported by [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], micro morphological characteristics were investigated using a light microscope (Olympus CX51, Japan).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMolecular characterization and phylogenetic identification of fungal isolates\u003c/h3\u003e\n\u003cp\u003eThe selected strain was sent to the Assiut University Mycological Centre (AUMC), Assiut University, Egypt, for extraction of DNA using the Solg\u0026trade; Genomic DNA Prep Kit (SolGent Co., Ltd, South Korea). PCR was conducted using ITS1(forward) (5\u0026prime;-TCC GTA GGT GAA CCT GCG G -3\u0026prime;), and ITS4 (reverse) (5\u0026prime;- TCC TCC GCT TAT TGA TAT GC -3\u0026prime;). The PCR products were purified with the SolGent PCR Purification Kit-Ultra and sequenced (SolGent Co., Ltd, South Korea). The BlastN technique was used to compare the acquired sequence of this fungus to that of the National Center for Biotechnology Information's (NCBI). For the phylogenetic analysis, sequence alignment was used MegAlign software (version 5.05)[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eBiosynthesis and characterization of CuONPs\u003c/h3\u003e\n\u003cp\u003e \u003cem\u003eL. aphanocladii\u003c/em\u003e mycelial disc (5 mm) was inoculated in 100 mL PDB at 28˚C for 7 days. Then, Whatman filter paper no. 1 was employed for the filtration procedure to yield cell-free filtrates. The filtrate was further purified through centrifugation at 6000 rpm for 5 minutes, at 4 ˚C. The fungal filtrate was examined for the preparation of CuONPs as documented by [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. In summary, 30 mL of CuSO\u003csub\u003e4\u003c/sub\u003e\u0026middot;5H\u003csub\u003e2\u003c/sub\u003eO (0.1 M) was combined with 10 mL of fungal filtrate in a flask, followed by stirring and heating the solution at 90˚C for 5 h. Following the incubation period, the reduction of copper ions was monitored visually and documented as a dark brown, confirming the biosynthesis of CuONPs. The final product was subjected to continuous washing with distilled water, centrifugation at 8000 rpm, and drying in a vacuum oven. The resultant material was thereafter kept at 4 ˚C for investigation.\u003c/p\u003e \u003cp\u003eA preliminary validation of CuONP production via mycelial filtrate was evaluated utilizing a UV-visible spectrophotometer (Shimadzu, Kyoto, Japan; Dual Beam Spectrophotometer, UV-1800) over wavelengths from 200 to 800 nm for UV-vis spectroscopy analysis.\u003c/p\u003e \u003cp\u003eThe investigation utilizing Fourier-transform infrared (FTIR) spectroscopy was conducted with a Perkin-Elmer 1430 infrared spectrophotometer, USA; that features an IR Affinity-1 model. It has a resolution of 4 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003eand a wavelength ranges from 400 to 4000 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Prior to examination, the powdered CuONPs were dehydrated and subsequently combined with KBr to create pellets for examination [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. FTIR spectroscopy measures infrared radiation at certain wavelengths, revealing functional group interactions during reduction. In addition, the morphological properties of CuONPs were analyzed using transmission electron microscopy (TEM). A limited quantity of nanoparticle dispersion was deposited onto a carbon-coated copper grid to produce thin films of CuONPs. The samples underwent drying in a vacuum desiccator and were subsequently analyzed using a TEM microscope (JEOL JEM 1400; Japan). After that, X-ray diffractometer (Bruker Co.D8 Discover, Cu target, Wavelength 1.54A, 40 Kv 40 mA, Germany) were implemented.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn vitro\u003c/b\u003e \u003cb\u003eevaluation of the antifungal efficacy of CuONPs against\u003c/b\u003e \u003cb\u003eF. oxysporum\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cem\u003eF. oxysporum f.\u003c/em\u003e sp. \u003cem\u003elycopersici\u003c/em\u003e RCMB008001 was obtained from the Mycology Regional Center, Al-Azhar University, Cairo, Egypt. The pathogenicity test was then confirmed according to [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Various concentrations of CuONPs (25, 50, and 100 ppm), and Maxim XL fungicide used as positive control (3.5%) were assessed for their impact on the radial growth of \u003cem\u003eF. oxysporum\u003c/em\u003e, following the methodology established by [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. A 0.5 cm diameter disc containing actively growing mycelium from a 7-day-old fungal culture, was positioned at the center of each agar plate. The plates were incubated at 28\u0026deg;C\u0026thinsp;\u0026plusmn;\u0026thinsp;2 for a duration of 4 days. \u003cem\u003eF. oxysporum\u003c/em\u003e growth inhibition percentage was obtained using the subsequent equation:\u003c/p\u003e \u003cp\u003eInhibition of pathogen proliferation (%) is calculated using the formula: (control growth\u0026thinsp;\u0026minus;\u0026thinsp;treated growth) / (control growth) \u0026times; 100.\u003c/p\u003e\n\u003ch3\u003ePreparation of fungal inoculum\u003c/h3\u003e\n\u003cp\u003eTo evaluate the efficacy of CuONPs over Fusarium root rot, a spore suspension of \u003cem\u003eF. oxysporum\u003c/em\u003e was grown on PDB [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. The spore suspension was agitated and cultured at 160 rpm and 30\u0026deg;C until the media became turbid. The spore solution was further diluted with sterilized distilled water to achieve the target spore concentration of 1 X 106 conidia/ mL.\u003c/p\u003e \u003cp\u003eIn vivo experiments\u003c/p\u003e\n\u003ch3\u003eAnalysis of the antifungal properties of CuONPs in combat to Fusarium root rot\u003c/h3\u003e\n\u003cp\u003eEffectiveness of CuONPs in mitigating Fusarium rot induced by \u003cem\u003eF. oxysporum\u003c/em\u003e was evaluated utilizing potato cultivar (\u003cem\u003eSolanum tuberosum\u003c/em\u003e L.) called Caruso, originating from Germany and imported by Daltex Co., 42 Wadi Al Nile, Gazirat Mit Oqbah, Agouza, Giza Governorate, Egypt. Each treatment was replicated three times, with four to five plants per replicate, using 45 cm diameter plastic pots containing 12 kg of soil (clay: sand, 3:1 v/v), which underwent autoclaving twice for 30 min at 121\u0026deg;C. The physicochemical properties of the soil were as follows: P\u0026thinsp;=\u0026thinsp;2.4, N\u0026thinsp;=\u0026thinsp;0.7, K\u0026thinsp;=\u0026thinsp;34.3, Mg\u0026thinsp;=\u0026thinsp;23.2, Na\u0026thinsp;=\u0026thinsp;6.1, Ca\u0026thinsp;=\u0026thinsp;5.6, and Cl\u0026thinsp;=\u0026thinsp;6.69 mg/kg. The soil exhibited alkalinity (7.1) and electrical conductivity (3.5 mS/cm). The pots were irrigated daily with tap water until full germination occurred and 500 ml of a fungal spore suspension containing 1 \u0026times; 106 conidia/mL. On the seventh day after sowing, germinated seedlings were divided into three primary infected groups: a control (tap water), spraying (100 ml), watering (100 ml) and infected group (500 ml of a fungal spore suspension containing 1 \u0026times; 106 conidia/mL for one week). The infected group was further subdivided into three treatment groups that received infected group, foliar spraying (100 ml) and watering (100 ml) weekly with CuONPs (100 ppm). The experiment was arranged in a completely randomized design with three replicates. Plants were harvested after 60 days of growth for subsequent analysis. The shoot length, root length, fresh weight and dry weight were measured for estimating the potato growth performance. The weight of biomass (g) was ascertained by drying it in a vacuum oven (60\u0026deg;C/3 days).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003ePhysiological studies\u003c/h2\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003eEstimation of hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e)\u003c/h2\u003e \u003cp\u003e[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e] identified hydrogen peroxide absorbance at 390 nm, with an extinction coefficient of 0.28 \u0026micro;M\u003csup\u003e\u0026ndash;1\u003c/sup\u003e cm\u003csup\u003e\u0026ndash;1\u003c/sup\u003e. Leaf tissue weighing 200 mg was homogenized for 10 minutes in an ice bath with 1 mL of an extraction mixture comprising 1 M potassium iodide, 0.1% (w/v) trichloroacetic acid, and 10 mM potassium phosphate buffer at pH 5.7. Results were expressed as micromoles per gram of fresh weight.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e\n\u003ch3\u003eEstimation of malondialdehyde content (MDA)\u003c/h3\u003e\n\u003cp\u003eThe thiobarbituric acid (TBA) assay quantified malondialdehyde (MDA), a lipid peroxidation marker, at wavelengths of 532 and 600 nm, as described by [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Homogenize 0.5 g of freshly harvested leaves in 10 ml of 5% (w/v) TCA. The homogenate underwent centrifugation for 15 minutes at 4000 rpm. TBA at a concentration of 0.677% (w/v) was mixed with equal volumes of supernatant. Following a 15-minute exposure to a boiling water bath, the mixture rapidly cooled down for 10 minutes in cold water.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eAssessment of various non-enzymatic and enzymatic antioxidants\u003c/h2\u003e \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e \u003ch2\u003eAssessment of total antioxidant capacity (TAC)\u003c/h2\u003e \u003cp\u003eThe phosphomolybdenum method was employed to quantify total antioxidant capacity (TAC), with the sample's absorbance measured at 765 nm following cooling. The TAC reagent was prepared using sulfuric acid (0.6 M), ammonium molybdate tetrahydrate (4 mM), and sodium phosphate dibasic solution (28 mM) as described by Prieto et al. (1999). The ethanolic extract was mixed with TAC and heated for 90 minutes.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eAssessment of ascorbic acid content (ASA)\u003c/h2\u003e \u003cp\u003e[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]quantified the concentration of ascorbic acid (ASA) in leaf tissue using a 5% sulfosalicylic acid solution for extraction. The reaction includes leaf extract, 2% sodium molybdate, 0.15 N sulfuric acid, and 1.5 mM disodium hydrogen phosphate. Following a 45-minute exposure to a 60\u0026deg;C water bath, the mixture was subsequently cooled and subjected to centrifugation. Absorbance was measured at 660 nm. ASA was quantified in mg/g dry weight through the application of a calibration curve.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eAssessment of reduced glutathione level (GSH)\u003c/h2\u003e \u003cp\u003e[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e], revealed a reduction in GSH levels. A quantity of 0.1 g of freshly harvested leaves was homogenized in 5 ml of 3% w/v sulfosalicylic acid and subsequently centrifuged for 10 minutes at 10,000 rpm. In the reaction mixture, 0.5 ml of 0.5 mM potassium phosphate buffer (pH 7.0), 0.5 ml of tissue extract, and 50 \u0026micro;l of 3 mM DTNB (5,5\u0026prime;-Dithiobis(2-nitrobenzoic acid) were utilized. Appropriate quantities were utilized. Absorbance was measured at 412 nm using the GSH standard curve to estimate GSH concentration.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eAssessment of total phenolic content\u003c/h2\u003e \u003cp\u003eThe total phenolic content was calculated by measuring absorbance at 650 nm and comparing it to a standard curve derived from various gallic acid concentrations, as outlined by [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Following the extraction of 0.1 g of desiccated tissues in 10 mL of 95% ethanol, the supernatants were combined and adjusted to a final volume of 10 mL. Following the combination of 1 mL of the extract with 0.1 mL of Foline reagent and 1 mL of Na\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e, the resulting mixture was diluted to a final volume of 5 mL using distilled water.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eAssessment of peroxidase (POD), ascorbate peroxidase (APX), polyphenol oxidase (PPO) activity\u003c/h2\u003e \u003cp\u003eThe extraction process for the antioxidant enzyme matched to the guidelines established by [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. The technique employed to assess POD was that of [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. The activity of POD was noted at 470 nm, and calculated using a coefficient of extinction of 26.6 mM⁻\u0026sup1; cm⁻\u0026sup1;. The activity of APX was quantified utilizing the method established by [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. The absorbance at 290 nm diminished during the hydrogen peroxide-mediated oxidation of ascorbate. The procedure for quantifying PPO was delineated by [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. The enzyme activity was quantified as \u0026micro;M/g f.wt. min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Then, the absorbance was recorded at 420 nm utilizing an extinction coefficient of 26.40 M\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEvaluation of gene expression by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and RNA extraction.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eRNA from potato leaves was extracted utilizing the Qiagen RNase Mini Kit. A thermocycler (MJ Research, Inc., PTC-100TM Programmable Thermal Controller, USA) was utilized to synthesize 20 \u0026micro;l of complementary DNA (cDNA) by RNA reverse transcription. Following one hour of enzyme activation at 42\u0026deg;C, a five-minute inactivation phase at 95\u0026deg;C ensued. Conduct triplicate qRT-PCR utilizing Fermentas SYBR Green PCR Master Mix (USA). In each reaction, a 25 \u0026micro;l mixture of primer pairs (\u003cem\u003eCHI\u003c/em\u003e, \u003cem\u003ePIN2\u003c/em\u003e, \u003cem\u003eIbBBx24\u003c/em\u003e, and \u003cem\u003ePR-1b\u003c/em\u003e) as listed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e was employed. The reaction was conducted using the RotorGene 6000 (QIAGEN, ABI System, USA). The study utilized the ACTIN gene as a reference. The genes analyzed had their relative expression quantified and calculated utilizing the method of [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eParticular primers utilized in this research\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGene name\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAbbreviation\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eForward (F) and reverse (R) primer\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eReference gene\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eACTIN\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eF: 5\u0026prime;-GGTAACATTGTGCTCAGTGGTGG-3\u0026prime;\u003c/p\u003e \u003cp\u003eR: 5\u0026prime;-AACGACCTTAATCTTCATGCTGC-3\u0026prime;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePathogenesis related protein\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003ePR-1b\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eF: 5\u0026prime;-GGCATCCCGAGCACAAAAT-3\u0026prime;\u003c/p\u003e \u003cp\u003eR: 5\u0026prime;-CTGCACCGGAATGAATCAAGT-3\u0026prime;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBox-transcription factor\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eIbBBx24\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eF: 5\u0026prime;-AAACGCACAATTGGAGCCAC-3\u0026prime;\u003c/p\u003e \u003cp\u003eR: 5\u0026prime;-GTAGGTCATCAACGGCCCAA-3\u0026prime;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eProtease inhibitor\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003ePIN2\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eF: 5\u0026prime;-ATGAGCCCAAGGCAAATATGTAC-3\u0026prime;\u003c/p\u003e \u003cp\u003eR: 5\u0026prime;-GCCAATCCAGAAGATGGACAA-3\u0026prime;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eChalcone isomerase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eCHI\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eF: 5\u0026prime;-TGGTGGCCTAGACAACGATGAGTT-3\u0026prime;\u003c/p\u003e \u003cp\u003eR: TCACACTCCCAACTTGGTTTCCCT-3\u0026prime;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe findings were reported as the mean of three replicates, and the standard error (SEs) was computed. Two-way ANOVA was employed for statistical analysis to identify significant differences among treatments. Analyses were conducted using XLSTAT software (version 2014.5.03), with a significant threshold was set at p\u0026thinsp;\u0026le;\u0026thinsp;0.01.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eMorphological and molecular characterization and phylogenetic identification of\u003c/b\u003e \u003cb\u003eL. aphanocladii\u003c/b\u003e \u003cb\u003eisolate\u003c/b\u003e\u003c/p\u003e \u003cp\u003eMacroscopic characteristics revealed that they were white in color, high on the medium's surface, and had a reddish appearance at the bottom of the plates. Additionally, the microscopic structures of \u003cem\u003eL. aphanocladii\u003c/em\u003e revealed that the fungus produced conidia directly on the hypha and possessed cylindrical aphanophialides that carried phialo-conidium (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The sequences of \u003cem\u003eL. aphanocladii\u003c/em\u003e AUMC-16936 strain and deposited in Gene bank with accession No.PV549634 showed 99.14% \u0026minus;\u0026thinsp;100% similarity and 93% \u0026minus;\u0026thinsp;100% coverage with several strains of the same species, \u003cem\u003eTrichoderma asperellum\u003c/em\u003e, included as outgroup strain (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). \u003cem\u003eL. aphanocladii\u003c/em\u003e AUMC-16936 was deposited in the DDBJ GeneBank nucleotides sequence database with accession number (PV549634).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eMycosynthesis and characterization of CuONPs\u003c/h2\u003e \u003cp\u003eIn this study, CuONPs were successfully biosynthesized using the fungal filtrate of \u003cem\u003eL. aphanocladii\u003c/em\u003e. The fungal filtrate shifted from a yellowish to a dark brown color, signifying the biofabrication of nanoparticles. Subsequently, various characterization techniques were employed to verify the production of CuONPs. The characteristic peak of CuONPs was observed at 350 nm using UV-visible spectroscopic analysis, as illustrated in Fig.\u0026nbsp;(2). Additionally, nanoparticles were examined using TEM to reveal further morphological characteristics. The TEM analysis, as illustrated in Fig.\u0026nbsp;(3A), demonstrated the existence of rod-shaped particles with sizes varying from 15.24\u0026thinsp;\u0026plusmn;\u0026thinsp;4.19 nm. The sharp XRD peaks confirm the crystalline nature of the mycosynthesized CuONPs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). The narrow peak widths and high intensities indicate small particle size. Prominent diffraction peaks at 2θ\u0026thinsp;=\u0026thinsp;19.113\u0026deg;, 29.786\u0026deg;, 32.596\u0026deg;, 35.577\u0026deg;, 38.729\u0026deg;, 48.783\u0026deg;, 53.556\u0026deg;, 58.210\u0026deg;, 61.549\u0026deg;, 66.080\u0026deg;, and 68.052\u0026deg; correspond to the (110), (111), (111), (002), (202), (113), (020), (202), (311), (220), and (311) planes of monoclinic CuONPs (JCPDS card no. 01-007-2551).\u003c/p\u003e \u003cp\u003eAdditionally, Fig.\u0026nbsp;(3C) displays the FTIR spectra of the cell-free filtrate and the produced nanoparticles within the 400\u0026ndash;4000 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e range. The data obtained unequivocally demonstrated the emergence of a new band at 422 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in the spectrum of the produced nanoparticles, indicative of CuO nanoparticles. Nevertheless, there are no bands corresponding to any of these bands in the spectrum of the cell-free filtrate. The recorded results indicated that both spectra (for cell-free culture and created nanoparticles) displayed principal bands at 3432, 2932, 1638, 1372, and 1041 cm \u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, demonstrating the involvement of the cell-free \u003cem\u003eL. aphanocladii\u003c/em\u003e filtrate in nanoparticle production.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eextract.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eIn vitro\u003c/b\u003e \u003cb\u003eantifungal efficacy of CuONPs\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe antifungal activity of CuONPs was evaluated at three concentrations on PDA using the radial growth inhibition (RGI) assay. The antifungal activity of CuONPs exhibited higher effect than Maxim 4FS as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, and B. The radial growth of \u003cem\u003eF. oxysporum\u003c/em\u003e strain was recorded as 49\u0026thinsp;\u0026plusmn;\u0026thinsp;2.0, 26\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5, and 20.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3 mm, respectively, at concentrations of 25, 50, and 100 ppm in CuONPs supplemented media. The percentages of \u003cem\u003eF. oxysporum\u003c/em\u003e growth suppression were recorded at 39%, 68%, and 75%, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eIn vivo\u003c/b\u003e \u003cb\u003eimpact of CuONPs on growth parameters of potato plants\u003c/b\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eImpact of CuONPs on plant height, fresh, and dry masses\u003c/h2\u003e \u003cp\u003eThis study assessed the efficacy of CuONPs in controlling soil-borne Fusarium wilt disease in potatoes by a pot bioassay. Three months post-seeding, the impact of CuONPs on potato was assessed regarding plant lengths, as well as fresh and dry weights (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). The findings demonstrate a notable enhancement in plant growth metrics following the exposure of potato plants to 100 ppm of CuONPs, administered either via spraying (SN) or watering (WN), resulting in increases of 15% and 10%, respectively, compared to the control treatment. In terms of fresh and dry biomasses, potato plants treated with CuONPs exhibited a notable enhancement, with increases of 55% and 113% in fresh weight, respectively, and 67% and 150% in dry weight, respectively, as compared to the negative control in healthy plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB, C).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eImpact of CuONPs on oxidative stress\u003c/h2\u003e \u003cp\u003eFigure (7) illustrates that a concentration of 100 ppm of CuONPs did not induce oxidative stress in potato plants, as there was no significant elevation in H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and MDA levels in the CuONP-treated plants compared to the negative control. Conversely, potato plants infected with \u003cem\u003eF. oxysporum\u003c/em\u003e exhibited a significant 29% and 51.8%increase in H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and MDA content, respectively, relative to the negative control group. Furthermore, treatment with CuONPs (SN and WN) effectively mitigated the oxidative stress caused by Fusarium infection, resulting in a reduction of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and MDA levels by 14% and 15%, 38.7 and 34.5% respectively, compared to the infected potato plants.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eImpact of CuONPs on antioxidant compounds\u003c/h2\u003e \u003cp\u003eThe current investigation demonstrated a considerable increase in total antioxidant capacity (TAC) in Fusarium-infected plants following the administration of CuONPs, with enhancements of 68% and 48% observed through spraying and watering, respectively, compared to infected plants alone. Conversely, infection with \u003cem\u003eF. oxysporum\u003c/em\u003e resulted in a substantial reduction in total antioxidant capacity (TAC) by 30% relative to uninfected potato plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA). The application of CuONPs, both singly (SN and WN), resulted in a substantial increase in total phenolic (TP) content by 88% and 60%, respectively, as compared to the negative control plants. A notable enhancement in TP content of infected plants treated with CuONPs was recorded, with increases of 194% and 145% attributed to the application of SN and WN, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eThe utilization of SN or WN markedly increased the levels of GSH and ASA in 60-day-old diseased potato plants. The enhancement ratios were 393% and 345% for GSH, and 39% and 34% for ASA, in comparison to infected plants. Infection with \u003cem\u003eF. oxysporum\u003c/em\u003e led to a substantial reduction in the levels of GSH and ASA in 60-day-old potato plants, decreasing by 71% and 21%, respectively, compared to the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC, D).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eImpact of CuONPs on enzymatic activity\u003c/h2\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e illustrates that infection with \u003cem\u003eF. oxysporum\u003c/em\u003e reduced the activities of POD, PPO, and APX in 60-day-old potato plants by 28%, 7%, and 75%, respectively, in comparison to healthy plants. In contrast, CuONPs administered via either SN or WN markedly improved the enzyme levels, approaching those observed in healthy plants. The maximum value was seen at APX enzyme, with CuONPs considerably enhancing enzyme activity by 825% and 600% for spraying and watering, respectively, in comparison to infected plants. Moreover, the utilization of SN or WN markedly enhanced the activity of POD and PPO in infected plants by 40% and 29% for POD, and 22% and 19% for PPO, respectively.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eInfluence of CuONPs on the relative gene expression levels of\u003c/b\u003e \u003cb\u003ePR1b, PIN2, IbBBx24\u003c/b\u003e, \u003cb\u003eand\u003c/b\u003e \u003cb\u003eCHI\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAs illustrated in Figure (10), potato plants infected with Fusarium and treated with CuONPs (SN and WN) exhibited elevated levels of \u003cem\u003ePR1b, IbBBx24\u003c/em\u003e, and \u003cem\u003eCHI\u003c/em\u003e in comparison to other treatments. Nonetheless, \u003cem\u003ePIN2\u003c/em\u003e in infected plants exhibited a significant increase when compared to other treatments. Interestingly, the expression of the \u003cem\u003ePIN2\u003c/em\u003e gene was not affected by CuONPs treatments, whether applied through foliar techniques or watering.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003ePearson Correlation Coefficient\u003c/h2\u003e \u003cp\u003ePearson's simple correlation provided a comprehensive visualization of the interrelationships among physiological, biochemical traits and molecular approaches in plants observed in various treatments with mycosynthesized CuONPs applications (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003e). The findings revealed strong positive correlations between growth parameters, in addition, phenolic content, and TAC. Growth parameters were also found to be positively related with augmented enzymatic antioxidants (PPO, POD, and, APX). Furthermore, the expression levels of \u003cem\u003ePR1b, IbBBx24\u003c/em\u003e, and \u003cem\u003eCHI\u003c/em\u003e showed positive correlations with (PPO, POD, and, APX), phenolic content, ASA, and GSH. Conversely, oxidative stress markers (H₂O₂, MDA) were showed strong negative correlations with growth parameters and antioxidants, indicating that increased oxidative damage is associated with reduced plant growth and metabolic activity. The positive correlation between H₂O₂ and \u003cem\u003ePIN2\u003c/em\u003e suggests that hydrogen peroxide may act as a signaling molecule inducing stress-related gene expression. Overall, the heat map not only visualizes the complex interplay between growth, antioxidants, and stress markers but also provides a foundation for understanding how plants balance growth and stress responses at the biochemical level. Such insights are crucial for developing strategies to enhance plant resilience in challenging environments.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003ePrincipal component analysis (PCA)\u003c/h2\u003e \u003cp\u003eIn comparison to the CuONPs treatments, the PCA results linked the gene expression data with physiological factors (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003e). According to the study's findings, 87.11% of the variation was explained by the first four PCs with eigenvalues greater than 1. PC1 was the most significant, accounting for 65.21% of the overall variation. GSH, ASA, phenolic, PPO, APX, and \u003cem\u003eCHI\u003c/em\u003e were the metrics that showed the strongest connection with the PC1, in decreasing order. PC2, which was responsible for 21.90% of the overall change, was most impacted by \u003cem\u003eIbbx24, PR-1b\u003c/em\u003e, fresh weight, dry weight, POD, shoot length, and TAC, in decreasing order. Significant correlations were found between PC3, which explained 12.89% of the variation, and adverse impact factors, MDA, \u003cem\u003ePIN2\u003c/em\u003e, and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. Additionally, CUONPs were found to favour growth, physiological characteristics, and gene expression, according to PCA. Nonetheless, there was a strong correlation between the contaminated potato and MDA, \u003cem\u003ePIN2\u003c/em\u003e, and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe search for sustainable bio-manufacturing platforms has positioned fungi as promising candidates for the green synthesis of nanomaterials. In this context, our study successfully demonstrates, for the first time, the metabolic potential of \u003cem\u003eLecanicillium aphanocladii\u003c/em\u003e AUMC 16936 to function as a microbial cell factory for the production of CuONPs, and establishes their potent efficacy against Fusarium wilt in potato plants. The biosynthetic process was visually indicated by a distinct color change of the reaction mixture from pale yellow to dark brown hue, indicating its role as a reducing and capping agent that converts copper sulfate into CuONPs and stabilizes them in colloidal sate. Biologically active metabolites derived from the fungal extract function as a capping agent, inhibiting nanoparticle agglomeration and modifying their biological activity [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. The analysis of the UV-visible spectra revealed an obvious absorption peak at around 350 nm, indicating the successful production of CuONPs by the tested fungus. The peak of CuO's UV absorption typically exhibits an absorption range between 280 nm and 360 nm [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Moreover, the absorption peak of excited CuONPs seen at 350 nm signifies a monodispersed sizes are distributed within the overall mixture [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. In order to gain additional understanding of the structural characteristics of the mycosynthesized CuONPs, the particles were examined using TEM. The nanostructured particles displayed a rod-shape with diameters ranging from 15.24\u0026thinsp;\u0026plusmn;\u0026thinsp;4.19 nm. This outcome parallels findings in earlier studies regarding the shape of CuNPs and CuONPs [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. Furthermore, the monoclinic structure of the mycosynthesized CuONPs was indicated by the XRD analysis, which showed diffraction peaks that matched the standard CuO.\u003c/p\u003e \u003cp\u003eFTIR spectroscopy analysis was conducted to validate the production of nanoparticles and the examination of their interactions the produced nanoparticles and active metabolites from the cell-free filtrate of \u003cem\u003eL. aphanocladii\u003c/em\u003e. The results clearly demonstrated the effective synthesis of CuONPs. The distinctive peak of CuO was detected at 422 cm⁻\u0026sup1;. These findings align with numerous investigations that documented peaks at wavenumbers between 500\u0026ndash;700 cm\u003csup\u003e\u0026ndash;1\u003c/sup\u003e, indicating the production of CuO [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. The data indicated that the prominent broad absorption band between 2500 and 3750 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e corresponds to the O\u0026ndash;H stretching of hydroxyl groups in the compound, while the peak at 2095.94 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e is attributed to the alkene (C\u0026thinsp;=\u0026thinsp;C), and the peak at 1638.94 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e may be ascribed to the amine group. During The peak at 1554.75 results from the C\u0026thinsp;=\u0026thinsp;C stretching of the aromatic group in tertiary amides, tannins, saponins, flavonoids, and terpenoids. The peak at 1372.40 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e may correspond to C-N stretching in aliphatic amines, while the peak at 1041.47 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e is related with C-O stretching in phenols, alcohols, esters, or, ethers and may also indicate C-N stretching in aromatic amines [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e, \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. The existence of several functional groups in the biomass of fungal filtrate indicates their significance in the reduction of metal precursors to produce CuONPs, then capped to enhance their stability.\u003c/p\u003e \u003cp\u003eNumerous widely available antifungal had minimal efficacy towards \u003cem\u003eFusarium\u003c/em\u003e spp. Consequently, significant focus is necessary to develop a biodegradable and efficient antifungal agent against \u003cem\u003eF. oxysporum\u003c/em\u003e [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. The antifungal effectiveness of CuONPs was estimated at three doses on PDA using the radial growth inhibition (RGI) assay, recognized as an effective and dependable approach for assessing the fungicidal properties of nanomaterials [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. Mechanistically, CuONPs demonstrate antifungal properties by generating reactive oxygen species (ROS) via Haber\u0026ndash;Weiss and Fenton-like processes, which compromise fungal cell walls by oxidizing chitin and glucan [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]. They also hinder respiratory metabolism and downregulate chitin synthase genes, resulting in morphological alterations. Furthermore, Cu\u003csup\u003e+\u003c/sup\u003e and OH\u003csup\u003e\u0026ndash;\u003c/sup\u003e ions engage with microbial proteins, resulting in the impairment of DNA, cell membranes, proteins, and lipids, finally leading to cellular death [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e, \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]. This potent direct toxicity suggests that \u003cem\u003eL. aphanocladii\u003c/em\u003e-derived CuONPs are viable candidates for replacing synthetic fungicides.\u003c/p\u003e \u003cp\u003eConsequently, to validate the agricultural utility of this bio-product, \u003cem\u003ein vivo\u003c/em\u003e trials revealed that application method plays a critical role in efficacy. Three months post-seeding, the impact of CuONPs on the growth parameters of potato was assessed. The findings demonstrate a notable enhancement in plant growth measurements following the exposure of potato to 100 ppm of CuONPs, administered either through spraying (SN) or watering (WN), compared to the control treatment. Our findings align with those of other nanoparticles, indicating that NPs enhance overall plant growth [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e, \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]. Thus, these results suggest that CuONPs can be utilized to promote plant growth and development. Furthermore, the application of CuONPs to infected plants (by spraying and watering) had a substantial stimulatory effect on the height of potato plants, as well as on their fresh and dry biomass, compared to the control group. It is suggested that, this increase in fresh weight may arise from the nanoparticles enhancing the functionality of photosystems I and II, as well as the redox state of plastoquinone within the electron transport chain [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOxidative stress induced by \u003cem\u003eF. oxysporum\u003c/em\u003e infection caused significant damage to plant cells, resulting in elevated levels of MDA and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e in the potato plants compared with those in the healthy control. These findings are in line with those of [\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e], who demonstrated that increasing MDA and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e levels in various potato varieties illustrate the impact of Fusarium wilt disease. Moreover, the results demonstrated that both CuONPs (SN and WN) displayed enhanced H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e scavenger activity, and MDA content, compared to the untreated control. Given the significance of copper as a microelement for plants, copper nanoparticles may therefore trigger defense systems in response to pathogen inoculation by producing extra proteins to stop pathogen entry or subsequent dissemination [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. These results are similar to those of [\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e], who reported that the application of copper nanoparticles under stress conditions resulted to decreasing of MDA and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e \u003cp\u003e \u003cem\u003eFusarium\u003c/em\u003e is a mycotoxins producing fungus, which induce ROS generation at the cellular level, potentially resulting in oxidative damage in plants [\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e]. To mitigate oxidative stress and defend against its detrimental effects, plant cells initiate antioxidant systems, encompassing the synthesis of both non-enzymatic and enzymatic substances [\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e]. The current investigation revealed that the treatments of CuONPs with \u003cem\u003eF. oxysporum\u003c/em\u003e enhanced the levels of secondary metabolites, including ascorbic acid, glutathione, total antioxidants, and total phenolic compounds, in comparison to infected plants. ASA and GSH molecules have a crucial antioxidant function and participate in various cellular signals within plant stress response pathways, including photosynthesis and hormone biosynthesis [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e, \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e]. Furthermore, Flavonoids and phenols are secondary metabolites, and their elevated levels are associated with the enhancement of induced resistance in plants, encompassing phytoalexins. They participate in hypersensitive responses, cell wall lignification, cellular apoptosis, and contribute to disease resistance [\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e, \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e]. Our findings align with those of Diana et al. [\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e], who demonstrated the efficacy of Graphene-Cu nanocomposite in enhancing resistance and eradicating \u003cem\u003eF. oxysporum\u003c/em\u003e infection in tomato plants by elevating levels of GSH, flavonoids, and other antioxidant enzymes.\u003c/p\u003e \u003cp\u003eThe antioxidant response in plants comprises many enzymatic molecules that remove reactive oxygen species (ROS) [\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e]. Peroxidases (APX, POD, and PPO) are recognized for their roles in regulating plant development, offering defense against diverse stresses, and facilitating the production of lignin; they bolster plant defense against diverse pathogens via strengthening cell wall barriers and communicating signals to adjacent healthy cells [\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e]. The results are consistent with those reported here, indicating that potato plants exhibit enhanced stress tolerance under these treatments. The application of CuONPs, whether through spraying or watering, markedly enhanced the activity of POD, PPO, and APX in 60-day-old infected potato plants relative to the infected controls. Similarly, our findings are corroborated by the research of Ashraf et al. [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e], which indicated that application of AgNPs enhanced phenolics contents and antioxidant enzyme activity(PAL, POD, and PPO) in tomato plants infected with \u003cem\u003eF. oxysporum\u003c/em\u003e. The preventive impact of CuONPs against Fusarium wilt disease in potato plants by enhancing both enzymatic and non-enzymatic defense mechanisms.\u003c/p\u003e \u003cp\u003eThe induction of this systemic resistance was further confirmed at the molecular level. The obtained results stated that the expression of the tested genes (\u003cem\u003ePR1b\u003c/em\u003e, \u003cem\u003eIbBBx24\u003c/em\u003e, and \u003cem\u003eCHI)\u003c/em\u003e upregulated significantly in response to CuONPs treatments, suggesting an increase in resistance mechanism of potato plants. The upregulation of pathogenesis related protein (PR1b), and chalcone isomerase (\u003cem\u003eCHI\u003c/em\u003e) triggers the salicylic acid-signaling pathway, and catalyzes the formation of flavonoids, respectively [\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e, \u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e]. This could be considered a systemic acquired resistance response. Moreover, B-box (BBX) family transcription factor IbBBX24 modulates the jasmonic acid (JA) pathway in sweet potato. IbBBX24 is crucial for the regulation of jasmonic acid production and signaling, enhancing resistance to Fusarium wilt and improving yield in sweet potato. Our results are in similar line with several reports addressed the role of these genes in enhancing pathogen resistance plants [\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e, \u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e]. Interestingly, \u003cem\u003ePIN2\u003c/em\u003e gene expression was upregulated significantly in infected plants with \u003cem\u003eF. oxysporum\u003c/em\u003e only. Moreover, there was no difference in the expression of this gene in infected plants treated with CuONPs compared to healthy plants. This might be due to the antifungal effect of CuONPs inside plant tissues, which led to normal expression rate of \u003cem\u003ePIN2\u003c/em\u003e gene compared to infected control plants.\u003c/p\u003e \u003cp\u003eIt is noteworthy that the mode of application significantly influenced efficacy, with foliar spraying consistently outperforming soil watering. This may be attributed to the fact that foliar-applied nanoparticles primarily penetrate the leaves through stomata and are subsequently conveyed through various plant tissues using symplastic and apoplastic pathways[\u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e, \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e]. Nutrients applied to the leaves are moved downwards and distributed to other plant parts. Likewise, the application of NPs on the leaves resulted in a higher NPs content within the root system, aiding nutrient absorption by root cells and enhancing their resistance to disease[\u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e83\u003c/span\u003e]. Thus, it can be posited that foliar application of nanoparticles augments the efficacy of plant protection strategies in comparison to conventional soil-root methods. Therefore, \u003cem\u003eL. aphanocladii\u003c/em\u003e AUMC-16936 serves as an efficient and eco-friendly microbial cell factory for the production of bioactive CuONPs inhibiting \u003cem\u003eF. oxysporum\u003c/em\u003e growth while also stimulating the potato plant's immune system via genetic and antioxidant pathways.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn conclusion, this study provides the first evidence utilizing \u003cem\u003eL. aphanocladii\u003c/em\u003e AUMC-16936 as a novel microbial cell factory for the green biosynthesis of CuONPs. The fungal bio-manufacturing process yielded highly stable, protein-capped nanorods. with dual-functional capabilities: direct fungicidal activity against \u003cem\u003eF. oxysporum\u003c/em\u003e and the ability to prime molecular defense mechanisms in potato plants. Foliar application of these biogenic nanoparticles proved to be the superior delivery strategy, significantly reducing disease severity and enhancing crop yield by upregulating key antioxidant and defense gene networks. By transforming a fungal metabolite stream into a high-value nano-agrochemical, this work bridges the gap between industrial biotechnology and sustainable agriculture. Future research should focus on optimizing fermentation conditions to scale up this mycosynthesis process, while also assessing their long-term environmental impacts such as soil accumulation and effects on non-target organisms.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eConsent for publication\u003c/h2\u003e \u003cp\u003eAll authors approved the manuscript. \u003cb\u003eCompeting interests\u003c/b\u003e The authors declare no competing interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding:\u003c/h2\u003e \u003cp\u003eThis research was funded by Princess Nourah bint Abdulrahman University Researchers Supporting Project number (PNURSP2026R318), Princess Nourah bint Abdulrahman University, Riyadh, Saudi Arabia.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eConceptualization: N.A.E, M.A.E, and D.E.E.; methodology: N.A.E, M.A.E, D.E.E. and M.H.; formal analysis and software: D.E.E. and M.H.; investigation: N.A.E, M.A.E, D.E.E. and M.H.; writing\u0026mdash;original draft preparation: D.E.E. and M.H; writing\u0026mdash;reviewing and editing: D.E.E., F.A.S., and M.H. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003ePrincess Nourah bint Abdulrahman University Researchers Supporting Project number (PNURSP2026R318), Princess Nourah bint Abdulrahman University, Riyadh, Saudi Arabia\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eAvailability of data and materials: Data is provided within the manuscript\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eDevaux A, Goffart J-P, Kromann P, Andrade-Piedra J, Polar V, Hareau G. The Potato of the Future: Opportunities and Challenges in Sustainable Agri-food Systems. Potato Res. 2021;64:681\u0026ndash;720.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBirch PRJ, Bryan GJ, Fenton B, Gilroy EM, Hein I, Jones JT, et al. Crops that feed the world 8: Potato: are the trends of increased global production sustainable? 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The use of copper as an antimicrobial agent in health care, including obstetrics and gynecology. Clin Microbiol Rev. 2019;32:10\u0026ndash;1128.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHajipour MJ, Fromm KM, Ashkarran AA, de Aberasturi DJ, de Larramendi IR, Rojo T, et al. Antibacterial properties of nanoparticles. Trends Biotechnol. 2012;30:499\u0026ndash;511.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAshraf H, Anjum T, Riaz S, Ahmad IS, Irudayaraj J, Javed S, et al. Inhibition mechanism of green-synthesized copper oxide nanoparticles from Cassia fistula towards Fusarium oxysporum by boosting growth and defense response in tomatoes. Environ Sci Nano. 2021;8:1729\u0026ndash;48.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSperdouli I, Adamakis I-DS, Dobrikova A, Apostolova E, Hanć A, Moustakas M. 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Graphene\u0026ndash;Cu Nanocomposites Induce Tolerance against Fusarium oxysporum, Increase Antioxidant Activity, and Decrease Stress in Tomato Plants. 2023.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMarslin G, Sheeba CJ, Franklin G. Nanoparticles alter secondary metabolism in plants via ROS burst. Front Plant Sci. 2017;8:832.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChandra S, Chakraborty N, Chakraborty A, Rai R, Bera B, Acharya K. Abiotic elicitor-mediated improvement of innate immunity in Camellia sinensis. J Plant Growth Regul. 2014;33:849\u0026ndash;59.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAl-Mokadem AZ, Alnaggar AE-AM, Mancy AG, Sofy AR, Sofy MR, Mohamed AKSH, et al. Foliar application of chitosan and phosphorus alleviate the potato virus Y-induced resistance by modulation of the reactive oxygen species, antioxidant defense system activity and gene expression in potato. Agronomy. 2022;12:3064.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNgaki MN, Louie GV, Philippe RN, Manning G, Pojer F, Bowman ME, et al. Evolution of the chalcone-isomerase fold from fatty-acid binding to stereospecific catalysis. Nature. 2012;485:530\u0026ndash;3.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhu J, Zhao W, Li R, Guo D, Li H, Wang Y, et al. Identification and Characterization of Chalcone Isomerase Genes Involved in Flavonoid Production in Dracaena cambodiana. Front Plant Sci. 2021;12:616396.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBanerjee K, Pramanik P, Maity A, Joshi DC, Wani SH, Krishnan P. Methods of using nanomaterials to plant systems and their delivery to plants (mode of entry, uptake, translocation, accumulation, biotransformation and barriers). Advances in phytonanotechnology. Elsevier; 2019. pp. 123\u0026ndash;52.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDjanaguiraman M, Anbazhagan V, Dhankher OP, Prasad PVV, Uptake. Translocation, Toxicity, and Impact of Nanoparticles on Plant Physiological Processes. Plants. 2024;13:3137.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHong J, Wang C, Wagner DC, Gardea-Torresdey JL, He F, Rico CM. Foliar application of nanoparticles: mechanisms of absorption, transfer, and multiple impacts. Environ Sci Nano. 2021;8:1196\u0026ndash;210.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"microbial-cell-factories","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"micf","sideBox":"Learn more about [Microbial Cell Factories](http://microbialcellfactories.biomedcentral.com/)","snPcode":"12934","submissionUrl":"https://submission.nature.com/new-submission/12934/3","title":"Microbial Cell Factories","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Mycosynthesis nanoparticles, Lecanicillium aphanocladii, Copper oxide, Fusarium oxysporum, Potato, Fausarium wilt","lastPublishedDoi":"10.21203/rs.3.rs-8360033/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8360033/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eUtilizing fungal metabolic processes for the eco-friendly synthesis of nanomaterials has arisen as a sustainable substitute for chemical manufacturing. To the best of our knowledge, this is the first report describing the mycosynthesis of bioactive CuONPs using \u003cem\u003eLecanicillium aphanocladii\u003c/em\u003e and evaluating their efficacy against potato Fusarium wilt.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eHerein, CuONPs were mycosynthesized utilizing the cell-free filtrate of \u003cem\u003eL. aphanocladii\u003c/em\u003e AUMC-16936, acting as a bio-reductant and capping agent. UV\u0026ndash;visible spectroscopy (UV\u0026ndash;Vis), Fourier transform infrared spectroscopy (FT-IR), Transmission electron microscopy (TEM), and X-ray spectroscopy (XRD) patterns confirmed the precise composition of the rod-shaped CuONPs with a mean particle size of 15.24\u0026thinsp;\u0026plusmn;\u0026thinsp;4.19 nm. The biogenic CuONPs demonstrated potent antifungal activity \u003cem\u003ein vitro\u003c/em\u003e against \u003cem\u003eFusarium oxysporum\u003c/em\u003e, with 100 ppm identified as the optimal inhibitory concentration. To validate the biological functionality of the synthesized particles, \u003cem\u003ein vivo\u003c/em\u003e trials were conducted on potato plants. Foliar application of the mycosynthesized CuONPs (100 ppm) proved superior to soil watering, significantly mitigating Fusarium wilt severity. The treatment triggered a systemic defense response, evidenced by the upregulation of antioxidant enzymes as ascorbate peroxidase (APX), peroxidase (POD), and polyphenol oxidase (PPO), along with non-enzymatic indicators including total antioxidant capacity (TAC), total phenolic content, ascorbic acid, as well as glutathione, leading to a marked reduction in oxidative stress markers (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, MDA). Moreover, elevated expression levels of several regulatory defense genes, such as \u003cem\u003ePR-1b\u003c/em\u003e, \u003cem\u003eIbBBx24\u003c/em\u003e, and \u003cem\u003eCHI\u003c/em\u003e, were noted in all plants that have been treated in comparison to infected ones.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003e \u003cem\u003eL. aphanocladii\u003c/em\u003e AUMC-16936 efficiently yields bioactive CuONPs that combat Fusarium wilt in potato plants offering a promising strategy for sustainable nano-fungicides.\u003c/p\u003e","manuscriptTitle":"Exploring the Potential of Lecanicillium aphanocladii AUMC-16936 as a Novel Microbial Cell Factory for the Biosynthesis of CuONPs Against Potato Fusarium Wilt","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-22 09:23:02","doi":"10.21203/rs.3.rs-8360033/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-01-09T13:23:02+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-09T13:22:39+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-09T03:26:59+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-06T07:03:24+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-31T06:01:13+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-27T00:45:07+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"290454608382255177713344722538536103749","date":"2025-12-24T00:54:57+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"317087333586845215239187443573835069716","date":"2025-12-19T10:29:28+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"227160688189677230218963435964605740152","date":"2025-12-19T05:18:59+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"15316016741815481536190500149768280802","date":"2025-12-19T04:16:40+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"44462043341407242447503385655087165019","date":"2025-12-18T21:03:06+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"325257523397687585229997483991795011197","date":"2025-12-18T16:29:23+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"113823912115172507182184128044939625327","date":"2025-12-18T15:24:57+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"293812743600063121582477625872904707131","date":"2025-12-18T15:07:52+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-12-18T14:54:07+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-12-16T22:10:48+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-12-16T08:22:35+00:00","index":"","fulltext":""},{"type":"submitted","content":"Microbial Cell Factories","date":"2025-12-14T19:23:13+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"microbial-cell-factories","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"micf","sideBox":"Learn more about [Microbial Cell Factories](http://microbialcellfactories.biomedcentral.com/)","snPcode":"12934","submissionUrl":"https://submission.nature.com/new-submission/12934/3","title":"Microbial Cell Factories","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a82b069c-9033-429b-967e-db4ba50cfdac","owner":[],"postedDate":"December 22nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-04-13T16:07:57+00:00","versionOfRecord":{"articleIdentity":"rs-8360033","link":"https://doi.org/10.1186/s12934-026-02970-7","journal":{"identity":"microbial-cell-factories","isVorOnly":false,"title":"Microbial Cell Factories"},"publishedOn":"2026-04-09 15:59:16","publishedOnDateReadable":"April 9th, 2026"},"versionCreatedAt":"2025-12-22 09:23:02","video":"","vorDoi":"10.1186/s12934-026-02970-7","vorDoiUrl":"https://doi.org/10.1186/s12934-026-02970-7","workflowStages":[]},"version":"v1","identity":"rs-8360033","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8360033","identity":"rs-8360033","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}